UC  NRLF 


B   H   EID   310 


-  be  'dnivcrsf ti5  of  Cbfcaso 

FOUNDED  BY  JOHN  D.  ROCKEFELLER. 


THE  BACTERIOLOGY  OF  WHOOPING- 
COUGH 


A  DISSERTATION 

SUBMITTED    TO      THE     FACULTY     OF     THE    OGDEN    GRADUATE    SCHOOL    OF 

SCIENCE    IN     CANDIDACY     FOR     THE     DEGREE     OF     DOCTOR 

OF    PHILOSOPHY. 

(department  of  pathology  AND  BACTERIOLOGY) 


BY 


DAVID  J.  DAVIS 


CHICAGO 

1906 


MEMCAL    .SCHOOL 


Digitized  by  the  Internet  Archive 

in  2007  with  funding  from 

IVIicrosoft  Corporation 


http://www.archive.org/details/bacteriologyofwhOOdavirich 


^be  mnlvcrslt^  ot  Cbfcaflo 

KOCNDED  BY  JOHN  D.  ROCKEFELLER, 


THE  BACTERIOLOGY  OF  WHOOPING- 
COUGH 


A  DISSERTATION 

SUBMITTED    TO      THE     FACULTY     OF     THE    OGDEN    GRADUATE    SCHOOL    OF 

SCIENCE    IN     CANDIDACY     FOR     THE     DEGREE     OF     DOCTOR 

OF    PHILOSOPHY. 

(department  of  pathology  and  bacteriology) 


BY 

DAVID  J.   DAVIS 


9     %  •      •      •  • 


CHICAGO 
1906 

1^ 


\^6(.  The 

Journal  of  Infectious 
Diseases 

FOUNDED  BY  THE  MEMORIAL  INSTITUTE  FOR  INFECTIOUS  DISEASES 


Vol.   3  March  2,  igo6  No. 


THE  BACTERIOLOGY  OF  WHOOPING-COUGH* 

David  J.  Davis. 

{^From  the  Memorial  Insiitute  for  Infectious  Diseases,  Chicago.) 

CONTENTS. 
I.  Introduction. 

Importance  of  Determining  the  Cause  of  Pertussis. 
Reasons  for  Assuming  it  an  Infectious  Disease. 
Possible  Nature  of  the  Virus. 
II.  Literature. 
Protozoa. 
Cocci. 
Bacilli. 
Conclusions  from  Literature. 

III.  Personal  Observations. 

Technique  and  Material. 

Bacteriology  of  Cases. 

Influenza-like  Organisms.  ^ 

Morphology. 

Cultural  Characteristics. 

Symbiosis. 

IV.  Animal  Experiments. 

Literature. 

Experiments  upon  Lower  Animals. 
Human  Inoculation. 
V.  Influenza-like  Organisms  in  Other  Diseases  and  in  Normal  Throats. 
Measles.    Influenza.    Meningitis.    Bronchitis.    Varicella.    Normal  Throats. 
Literature. 
Pertussoid. 
VI.  Nomenclature  of  the  Bacilli, 
VII.  Summary  and  Conclusions. 

♦This  work  was  made  possible  by  a  fund  given  by  Mrs.  F.  R.  Lillie  for  the  study  of  whooping-cough. 


19i-Sr9 


2  David  J.  Davis 

I.  INTRODUCTION. 

The  etiology  of  whooping-cough  has  not  as  yet  been  determined, 
notwithstanding  the  large  number  of  investigations  upon  this  sub- 
ject. The  disease  has  many  serious  aspects,  and  the  problem  of  its 
etiology  surely  deserves  to  be  ranked  among  the  most  important  of 
the  still  numerous  problems  concerning  infectious  diseases  that  so 
far  have  not  been  solved.  Like  many  other  infectious  diseases,  the 
therapeutics  of  whooping-cough  is  in  a  most  unsatisfactory  condition ; 
and  it  is  likely  to  remain  so,  at  least  until  the  etiology  is  determined. 
No  specific  is  known,  and  while  some  drugs  may  mitigate  the  symp- 
toms slightly,  none  has  any  appreciable  effect  upon  the  general  course 
of  the  disease.  As  regards  its  prevalence,  it  occupies  a  position  in 
the  first  ranks  of  the  infectious  diseases  of  children,  probably  being 
equaled  only  by  measles  in  this  respect.  While  in  strong,  healthy, 
and  older  children  the  disease  may  not  be  considered  very  serious, 
however,  owing  to  its  prolonged  course,  especially  in  the  winter 
months,  with  an  abundance  of  serious  comphcations  and  sequelae, 
it  must  be  looked  upon,  as  the  high  rate  of  mortaUty  abundantly  proves, 
as  one  of  the  most  dreaded  diseases  for  the  young  infants  and  the 
more  dehcate  children. 

Reasons  jor  assuming  it  an  injections  disease. — There  can  be  no 
doubt  that  whooping-cough  is  an  infectious,  transmissible  disease. 
Several  convincing  reasons  may  be  given  for  this.  Transmission  of 
the  disease  certainly  occurs  by  contact  between  children,  and  Baginsky 
states  positively  that  he  has  observed  transmission  by  a  third  person. 
There  is  no  doubt  that  the  virus  adheres  to  rooms  and  fomites.  The 
frequent  epidemic  character  of  the  disease,  and  its  endemic  character 
in  cities,  indicate  its  infectious  nature.  Again,  the  disease  has  a 
fairly  definite  period  of  incubation.  There  is  some  difference  of 
opinion  as  to  the  length  of  this  period,  but  most  observers  agree  that 
it  is  from  7  to  14  days.  Immunity  is  as  a  rule  conferred  by  the  attack. 
Indicative  of  its  infectious  nature  is  also  the  relatively  high  leucocyto- 
sis,  which  varies  from  12,000  to  45,000,  the  increase  in  the  mononu- 
clears being  particularly  characteristic.  Not  only  does  the  clinical 
course  of  the  disease  indicate  an  infectious  catarrhal  condition  of  the 
upper  respiratory  passages,  but  the  pathologic  anatomy  substantiates 
such  a  conclusion.     The  mucosa,  especially  of  the  larynx  and  trachea, 


Bacteriology  of  Whooping-Cough  3 

is  hyperemic  and  usually  covered  with  more  or  less  mucus.  According' 
to  Arnheim,  who  has  had  numerous  postmortems,  there  is  a  marked 
desquamative  catarrh  of  the  larynx  and  trachea,  with  swelling  of  the 
neighboring  lymph  glands.  We  know  also  that  the  posterior  laryngeal 
wall,  near  the  vocal  cords,  is  the  point  whence  severe  coughing  may 
be  most  readily  produced,  and  this  locality  is  supplied  by  the  superior 
laryngeal  nerve,  the  one  which  produces  cough  when  irritated.  This 
may  be  given  as  a  reason  for  viewing  the  disease  as  an  inflammatory 
irritation  of  the  laryngeal  mucosa ;  however,  the  possibility  of  reflex 
coughs  due  to  irritation  of  the  nasal  mucosa,  and  the  possibility  of  a 
toxic  irritation  of  the  coughing- center  in  the  medulla,  though  specula- 
tive in  character,  might  be  considered. 

The  above  facts,  therefore,  furnish  strong  evidence  that  the  disease 
is  due  to  some  form  of  a  zymotic  agent,  locaHzed  in  all  probabiHty 
in  the  upper  respiratory  passages,  particularly  the  larynx  and  trachea. 
This  is  important  in  determining  the  point  of  attack  in  attempting 
to  solve  the  problem  of  etiology. 

Possible  nature  of  the  virus. — From  what  is  known  concerning  the 
nature  of  the  causative  virus  of  diseases  of  an  infectious  character, 
we  may  consider  three  possible  agents,  viz. :  protozoa,  bacteria,  and 
enzymes.  The  last-mentioned  agent  may  be  dismissed  by  saying 
that,  in  the  animal  world  at  least,  there  does  not  exist  at  present 
evidence  to  indicate  that  such  substances  play  any  part  whatever  in 
the  primary  causation  of  any  known  infectious  diseases.  In  the  plant 
world,  however,  certain  recently  discovered  facts  seem  to  point  to 
the  possibility  of  enzymes  being  the  cause  of  certain  diseases  of  an 
infectious  nature;  and  if  this  be  true  we  should  guard  against  over- 
looking such  a  cause  in  studying  animal  diseases  of  unknown  etiology. 
Protozoa  and  bacteria  are  the  common  agents  in  the  production  of 
disease,  and  it  is  among  these  that  we  naturally  look  for  the  final 
solution  of  the  problem.  They  may  be  microscopic  or  ultramicro- 
scopic.  We  are  familiar  today  with  several  diseases  in  animals  which 
are  undoubtedly  due  to  ultramicroscopic  organisms.  Such  are  foot- 
and-mouth  disease,  hydrophobia,  and  probably  hog- cholera.  It  has 
been  demonstrated  that  these  can  be  produced  in  animals,  and  there- 
fore abundant  material  is  available  for  careful  experimental  work. 
Whooping-cough,  so  far  as  we  know,  does  not  occur,  and  cannot  be 


4  David  J.  Davis 

'produced,  in  animals,  and  we  are  therefore  handicapped  greatly  in 
not  being  able  to  apply  this  method  to  the  study  of  the  disease.  There 
is  Httle  doubt  but  that  its  cause,  as  well  as  that  of  many  other  human 
diseases  of  unknown  origin,  would  be  soon  cleared  up  if  only  available 
material  for  experimentation  could  be  procured.  The  possibihty, 
however,  of  an  etiologic  ultramicroscopic  organism  should  be  kept 
in  mind  in  studying  this  disease,  and  methods  devised  accordingly. 

II.  LITERATURE. 

The  literature  upon  the  bacteriology  of  pertussis  is  as  confusing 
as  it  is  extensive.  Almost  every  year  one  or  more  new  organisms 
are  described  and  assigned  an  etiologic  r61e  in  the  disease.  Curi- 
ously enough,  many  of  the  investigators  find  organisms  differing 
from  those  found  by  every  other  investigator,  and  their  enthusiasm 
has  not  infrequently  led  them  into  controversies  of  a  more  or  less 
personal  character.  After  going  over  the  literature  one  is  clearly 
impressed  with  the  idea  that  a  great  deal  of  rather  superficial  work 
has  been  done  on  the  subject,  and  very  little  that  is  really  thorough. 

For  convenience  it  may  be  well  to  divide  the  findings,  as  Jochmann 
and  Krause  have  done,  into  three  classes,  namely  protozoa,  cocci, 
and  bacilli,  and  discuss  each  briefly. 

Protozoa. — Henke'  in  1874  and  Deichler^  in  1886  found  constantly,  in  the  sputum 
of  whooping-cough  patients,  bodies  which  they  interpreted  as  protozoa.  Kurloff3  in 
1896  reported  similar  findings.  From  the  illustrations  accompanying  his  paper  one 
can  scarcely  draw  any  conclusion  other  than  that  they  are  desquamated,  ciliated 
epithelial  cells.  Behla4  in  1898,  who  also  found  ameba-like  bodies  in  fresh  pertussis 
sputum  and  assigned  to  them  an  etiological  significance,  interprets  Kurloflf's  ciliated 
bodies  as  epithelial  cells,  but  says  that  the  ameba-like  bodies,  which  Kurloflf  also 
described,  were  the  same  as  his  bodies.  All  of  the  above  results  were  inadequately 
controlled  and  only  indefinite  data  are  given  concerning  the  cases  examined.  The  inter 
pretations,  ■  for  the  most  part,  have  been  based  upon  observations  made  in  unstained 
specimens.  Behla  states  that  staining  was  of  no  differential  diagnostic  value.  The 
usual  interpretation  given  to  these  results  by  later  investigators  is  that  leucocytes 
and  epithelial  cells  have  been  n  istaken  for  protozoa. 

Cocci. — Certain  early  observations,  such  as  those  recorded  by  Moncorvo,  Barlow 
and  Broadbent,  Haushalter  and  Mircoli,  are  of  no  significance,  and  need  not  be  dis- 
cussed. Ritters  in  1892,  and  later  in  1896,  described  a  small  diplococcus  which  he 
obtained  constantly  from  146  cases  of  pertussis,  and  which  he  considered  the  probable 

'  Deutsches  Arch.  f.  klin.  Med.,  1874,  12,  p.  630.         3  Ceniralbl.  f.  BakL,  1896,  19,  p.  513. 

»  Bautngarten's  Jahresbericht,  1886,  2,  p.  347.  •♦  Deutsche    med.  Wchnschr.,  1898,  24,  p.  299' 

s  Berl.  klin.  Wchnschr.,  1892,  29,  p.  1276;  also  1896,  33:,  p.  1040. 


Bacteriology  of  Whooping-Cough  5 

cause.  His  results  could  not  be  verified  by  Cohn  and  Neumann  or  by  Czaplewski 
and  Hensel.  In  1899  his  assistant,  Buttermilch,'  attempted  to  identify  Ritter's 
organism  with  the  organism  described  by  Vincenzi  and  also  with  that  described  by 
Czaplewski  and  Hensel.  Neither  Ritter  nor  Buttermilch  used  blood  media,  so  that 
their  results  are  not  directly  comparable  with  those  obtained  by  later  investigators. 

Bacilli. — Burger^  in  1883  was  probably  the  first  to  call  attention  to  numerous 
bacilli  in  the  sputum  of  whooping-cough  patients.  He  made  no  culture  experiments, 
studying  only  stained  preparations.  He  called  attention  to  the  possibility  of  diagnos- 
ing the  disease  from  the  very  large  number  of  organisms  seen  in  the  sputum.  His 
results  are  of  little  value,  because  no  cultures  were  made.  Afanassiew,3  in  1887, 
described  short  motile,  bacilli,  often  in  masses  and  chains.  He  claims  that  he  was 
able  to  produce  the  disease  in  dogs  and  rabbits  by  the  injection  of  pure  cultures  of  the 
bacillus.  He  studied  10  cases.  He  did  not  use  blood  media,  and  his  method  of 
obtaining  pure  cultures  is  questionable.  Von  Genser,  Wendt,  and  Szemetschenko 
claim  to  have  substantiated  his  results. 

Spengler4  was  apparently  the  first  to  describe  an  organism,  in  pertussis  sputum, 
closely  resembling  the  influenza  bacillus  in  its  morphological  and  biological  aspects. 
His  organisms  are  somewhat  thicker  and  larger  than  the  influenza  bacillus,  and  have 
often  a  tendency  to  thread  formation.  Sometimes  the  protoplasm  of  the  cells  is  filled 
with  them.  They  grow  only  upon  hemoglobin  media.  He  considered  his  organism 
of  etiological  importance  in  the  disease. 

Kopliks  found  a  small,  short,  motile  bacillus  in  smears  and  culture  in  the  sputum 
in  13  out  of  16  cases  investigated.  It  grew  on  ordinary  media,  but  throve  much  better 
when  hydrocele  fluid  was  added.  It  was  pathogenic  to  white  mice,  and  intravenous 
injections  into  rabbits  caused  pyemia.  It  is  very  doubtful  whether  he  was  working 
with  pure  cultures,  as  his  method  of  isolating  the  organism  was  hardly  reUable.  His 
results  with  animals  suggest  the  presence  of  some  of  the  ordinary  pus  producers  of  the 
mouth  and  throat. 

Czaplewski  and  Hensel*^  likewise  found  a  small,  short,  polar-staining  bacillus, 
slightly  larger  than  the  influenza  bacillus,  but  which  grew  upon  non-hemoglobin 
media.  They  isolated  the  organism  from  sputum  on  blood-serum  plates.  Morpho- 
logically it  resembled  very  closely  the  influenza-like  organisms  described  by  Spengler, 
Jochmann,  and  Vincenzi.  Buttermilch  contended  that  it  was  identical  with  Ritter's 
diplococcus.  Spengler  said  that  it  was  the  same  as  his  bacillus,  but  that  Czaplewski 
and  Hensel  did  not  obtain  it  in  pure  culture,  and  hence,  when  transferred  to  other 
media,  the  mixed  organisms  developed  which  were  mistaken  for  the  bacilli.  Czaplew- 
ski's  results  were  confirmed  independently  by  Zusch,7  who  used  very  much  the  same 
technique,  and  also  by  A.  Cavasse.  The  latter,^  in  1904,  described  another  organism, 
motile,  growing  only  in  liquids,  staining  very  poorly,  and  never  before  observed  in 
pertussis  sputum;  its  significance  he  does  not  attempt  to  indicate.  Vincenzi,?  in  18 
cases,  found  a  small,  influenza-like  organism,  non-Gram  staining,  which  grew  on  non- 
hemoglobin  media.     He  says  that  it  is  different  from  Czaplewski's  organism. 

Elmassiani  described  an  organism  identical  with  Pfeiffer's  influenza  organism, 

'  Ibid.,  iSgg,  36,  p.  367.  »  Ibid.,  1883,  9,  p.  7. 

»  St.  Petersburger  med.  Wchnschr.,  1887,  4,  p.  349. 

*  Deutsche  med.  Wchnschr.,  1897,  23,  p.  830.  i  Centralbl.  /.  Bakt.,  1898,  24,  pp.  721,  769. 

» Centralbl.  /.  Bakt.,  1897,  22,  p.  222.  ^  Arch.  gin.  de  Mid.,  1904,  193,  p.  1345- 

^Deutsche  med.  Wchnschr.,  1897,  23,  p.  586.  <>  Deutsche  med.  Wchnschr.,  1898,  24,  p.  631. 


6  David  J.  Davis 

except  that  it  grows  on  serum  media.  He  found  it  in  eight  out  of  32  cases  of  whooping- 
cough,  and  also  obtained  it  from  cases  of  acute  bronchitis,  pulmonary  tuberculosis, 
and  pneumonia.  He  thinks  it  very  probable  that  it  is  the  same  as  Pfeiffer's  bacillus, 
which  he  Hkewise  tested,  and  found  that  it  also  would  grow  upon  his  serum  agar.  He 
does  not  consider  it  proved  that  influenza  is  caused  by  Pfeiffer's  organism.  Animal 
experiments  were  for  the  most  part  negative.  He  questioned  its  etiological  significance 
for  whooping-cough.  There  can  be  little  doubt  that  this  organism  is  the  same  as  that 
described  by  Spengler,  and  later  by  Jochmann  and  Krause. 

Luzzatto's  Bacillus  minutissimus  sputi^  corresponds  to  Elmassian's  bacillus  in 
practically  every  respect.  He  studied  41  cases.  He  also  described  an  organism 
resembling  the  pneumococcus. 

Arnheim,3  in  1900,  using  Czaplewski's  technique,  found  Czaplewski  and  Hensel's 
organism  both  in  sputum  of  patients  and  in  two  out  of  three  autopsies.  In  1903  he 
reported  his  findings  in  eight  autopsies.  He  claims  to  have  cultivated  the  organism 
from  the  lung  tissue  and  tracheal  mucus.  In  sections  he  observed  the  bacteria  espe- 
cially in  the  wall  of  the  trachea,  in  the  lungs,  and  in  great  numbers  in  the  large  des- 
quamated epithelial  cells.  The  organisms  grow  on  ordinary  media,  and  his  cultures 
show  very  irregular  coccus  forms,  thread  forms,  with  enlarged  ends,  and  various 
involution  forms.  With  Gram's  method  of  staining,  he  says  that  some  of  the  organisms 
stain  and  some  do  not. 

Jochmann  and  Krause,4  in  190 1,  found  influenza-Hke  bacilli  in  sputum  of  pertussis 
cases  which  belonged  to  three  distinct  classes  (A,  B,  C)  as  determined  by  their  reac- 
tion to  Gram's  stain  and  by  their  biological  properties.  In  18  out  of  31  cases,  among 
which  were  three  autopsies,  were  found  small,  non-Gram-staining,  influenza-like 
bacilli  (Class  A),  which  grew  only  in  the  presence  of  hemoglobin.  This  organism 
they  called  Bacillus  pertussis  Eppendorf.  In  four  cases  they  found  similar  bacilli 
(Class  B),  which,  however,  grew  on  hemoglobin-free  media.  They  considered  this 
organism  the  same  as  that  described  by  Czaplewski  and  Hensel,  and  thought,  because 
so  infrequently  found,  that  it  could  not  be  the  cause  of  pertussis.  In  three  cases  they 
found  a  Gram-staining  bacillus  (Class  C)  growing  without  the  presence  of  hemoglobin. 
The  occurrence  of  these  various  forms  in  pertussis,  they  think,  explains  the  discordant 
results  of  the  previous  investigations.  In  1903  JochmannS  reports  finding  the  B. 
pertussis  Eppendorf  altogether  in  60  cases  and  in  23  autopsies.  He  does  not  report 
further  findings  of  organisms  belonging  to  the  Classes  B  and  C.  He  says  that  one 
has  as  much  right  to  consider  this  bacillus  the  cause  of  whooping-cough  as  to  consider 
the  influenza  bacillus  the  cause  of  influenza. 

In  1903,  Reyher^  reports  the  constant  presence  of  Czaplewski's  "  Polbacterium  " 
in  34  cases  of  pertussis.  He  found  them  in  sputum,  once  in  pus  from  the  ear,  some- 
times in  the  nasal  secretion,  in  the  mucus  of  the  larynx  and  trachea  of  nine  autopsies, 
and  in  one  instance  saw  them  in  sections  of  the  larynx  and  trachea  in  the  epithelial 
cells.  He  states  that  the  organism  destains  by  Gram.  He  cannot  confirm  Joch- 
mann's  work.     His  report  in  many  respects  is  very  indefinite. 

In  1903  Neisser,!  from  conjunctivitis  in  measles,  obtained  B.  xerosis  and  an  influ- 
enza organism  which  he  cultivated  together  for  20  generations  on  plain  agar.  He  tested 
strains  from  pertussis,  scarlet  fever,  and  measles,  and  could  not  detect  any  difi'erence 

»  Ann.  de  I'lnst.  Past.,  1899,  13,  p.  621.  »  Centralbl.  f.  Baki.,  1900,  27,  p.  817. 

3  Berl.  klin.  Wchnschr.,  1900,  37,  p.  703;  also  Virchow  Archiv,  1903,  174,  p.  530. 

*Ztschr.,  j.  Hyg.,  1901,  36,  p.  193.  s  Ibid.,  1903,  44,  p.  513. 

^  Jahrbtich  f.  Kindcrh.,  1903,  58,  p.  605. 


Bacteriology  of  Whooping- Cough  7 

morphologically,  culturally,  or  by  immunization  experiments.  They  grew  well  through 
many  generations  with  Uving  B.  xerosis,  but  not  with  the  dead  organisms  or  their 
extracts. 

Manicatide*  found  a  short,  thick,  Gram-staining  bacillus  capable  of  growing  on 
ordinary  media  in  67  out  of  80  cases,  and  says  it  is  different  from  any  organism  pre- 
viously described.  Animal  experiments  gave  negative  results.  He  immunized  three 
sheep  and  two  horses,  and  obtained  a  distinct  agglutination  at  a  dilution  of  1:32. 
He  treated  89  cases  with  the  immune  serum,  and  claims  that  it  cures  cases  not  over  15 
days  old  in  from  2  to  12  days.  For  the  above  reasons,  he  considers  this  germ  the 
specific  cause  of  the  disease. 

Smit3  found,  in  24  cases,  Jochmann's  B.  pertussis  Eppcndorf  constantly.  Neither 
blood  serum  of  patients  nor  the  serum  of  a  horse  or  goats  immunized  to  this  organism 
had  any  agglutinative  properties.  In  rabbits,  by  inoculation  with  this  organism,  he 
obtained  lameness  and  a  general  infection,  and  also  produced  a  conjunctivitis  with  the 
bacilH  in  the  exudate.  He  treated  nine  patients  with  immunized  horse  serum  with  no 
perceptible  effect.  In  18  cases  he  failed  to  find  Manicatide's  bacillus  in  a  single 
instance.  With  the  bacillus  which  he  obtained  from  Manicatide,  he  obtained  some 
agglutination  with  the  blood  serum  of  pertussis  patients.  He  does  not  consider  either 
Jochmann's  bacillus  or  Manicatide's  bacillus  as  the  cause  of  the  disease. 

Very  recently  Martha  Wollstein4  isolated  from  29  out  of  30  cases  a  bacillus  identi- 
cal with  that  described  by  Spengler  and  Jochmann.  This  organism  was  agglutinated 
by  serum  of  pertussis  patients  at  dilutions  of  i :  200,  and  sometimes  at  i :  500,  With 
normal  serum,  the  tests  all  gave  negative  results  above  dilutions  of  1:10.  With  influ- 
enza bacilli,  the  serum  of  pertussis  patients  did  not  react  in  dilutions  higher  that  i :  20, 
Serum  from  rabbits  immunized  with  the  pertussis  organism  gave  positive  agglutina- 
tion tests  with  the  same  organism  at  dilutions  of  1:500,  with  the  influenza  organism 
at  1 :  200.  Serum  from  animals  immunized  with  influenza  bacilli  agglutinated  the 
influenza  organism  at  i :  200  and  the  pertussis  organism  at  i :  50.  Absorption  tests 
with  immunized  sera  also  showed  marked  reduction  in  agglutinins  after  a  saturation 
with  the  bacilli.  She  concludes  that  the  pertussis  organism  belongs  to  the  influenza 
group,  but  can  be  differentiated  from  B.  infltienzae  by  agglutination  reactions  with  the 
blood  of  patients  and  immune  animals.  Morphologically  it  is  slightly  larger  than  the 
influenza  bacillus. 

Conclusions  jrom  literature. — In  summing  up  the  literature  on  per- 
tussis the  most  striking  feature  is  the  lack  of  unity  in  the  results.  The 
work  of  the  earlier  investigators  is  of  little  value,  because  bacterio- 
logical technique  at  that  time  was  so  Httle  developed.  Undoubtedly 
the  chief  reason  for  the  diversity  of  results  lies  in  the  variety  of  the 
methods  employed.  Lack  of  knowledge  of  the  normal  flora  of  mouth 
and  throat  also  has  probably  had  much  to  do  with  the  variety  of  results 
obtained.  Many  of  the  investigators  have  not  at  all  appreciated  the 
importance  of  careful  control  of  their  results.     These  facts,  together 

•  Deutsche  med.  Wchnschr.,  1903,  29,  p.  462.  •  Zlschr.  }.  Hyg.,  1004,  45.  P-  469. 
3  Bacteriologische  Onderzoekingen  bij  Kinkhoest,  Thesis,  Amsterdam,  1905. 

*  Jour,  of  Exper.  Med.,  1905,  7.  P-  335 


8  David  J.  Davis 

with  an  evident  attempt  on  the  part  of  some  of  the  investigators  to 
exaggerate  minor  differences  in  order  to  be  able  to  announce  their 
organism  as  "heretofore  undescribed,"  have  been  responsible  for  the 
resulting  chaotic  mass  of  data. 

However,  there  has  been  found  with  some  degree  of  constancy, 
in  the  sputum  of  patients,  a  small,  short  bacillus,  often  described 
as  influenza-Hke,  both  in  smears  and  in  culture ;  and  it  may  be  further 
stated  that,  with  few  exceptions,  it  has  been  a  Gram-negative  organ- 
ism.    Further  than  this  one  finds  Httle  harmony  in  the  results.     The 
organism  described  by  Spengler  and  by  Jochmann  and  Krause  grew 
only   on   hemoglobin   media.     Those   described   by   Luzzatto   and 
Elmassian  grew  in  human  serous  fluids,  and  as  these  fluids  often 
contain  small  amounts  of  hemoglobin,  these  latter  organisms  are 
probably  the  same  as  those  described  by  Spengler  and  by  Jochmann 
and  Krause.     Jochmann  considers  his  organism  different  from  that 
described  by  Spengler  because  of  certain  minor  morphological  differ- 
ences.    There  is  no  doubt  but  that  both  are  dealing  with  the  same 
organism,  as  Spengler  has  very  emphatically  contended.     The  later 
findings  of  Smit  and  Wollstein  strongly  emphasize  the  significance 
of  this   organism.     Just   what   organism   Czaplewski   and   Hensel, 
Arnheim,  Reyher,  and  some  others,  have  been  dealing  with  is  very 
difficult  to  say.     After  reading  their  articles  one  is  inclined  to  question, 
as  Spengler  did,  whether  or  not  they  were  dealing  with  a  pure  culture. 
Their  varying  reports  concerning  its  reaction  to  Gram's  stain,  its 
morphology  (Arnheim),  and  the  questionable  methods  used  in  obtain- 
ing pure  cultures,  make  one  suspicious  of  the  whole  work.     It  is 
probable  that  they  have  been  dealing  with  the  same  organism  as  that 
which  Spengler  and  Jochmann  described.     This  organism  will  grow, 
for  a  few  generations  at  least,  on  non-hemoglobin  media  if  another 
organism  is  present  with  which  it  may  grow  in  symbiosis.     There- 
fore, if  sputum  is  smeared  on  such  media  as  Czaplewski  used,  the 
Spengler- Jochmann  organism  will  grow  because  there  are  always 
other  organisms  present.     But  it  is  a  difficult  matter  to  obtain  it  in 
pure  culture,  and  Czaplewski  and  Reyher  speak  of  the  trouble  they 
had  in  this  respect. 

No  one  has  found  an  organism,  either  before  or  since  Manicatide's 
report,  that  corresponds  to  that  described  by  him.     This  is  all  the 


Bacteriology  of  Whooping- Cough  9 

more  remarkable,  since  his  bacillus  grows  readily  on  all  ordinary 
media.  He  very  kindly  sent  ;ne  a  culture  of  his  organism,  which 
I  was  thus  able  to  compare  with  the  various  organisms  found  in 
pertussis  sputa  of  the  cases  reported  in  this  paper,  and  in  not  one 
instance  have  I  been  able  to  obtain  an  identical  organism.  It  is  an 
entirely  different  organism  from  the  influenza-like  bacillus. 

Summing  up,  one  seems  justified  in  concluding  that,  with  the 
exception  of  Manicatide,  probably  all  of  the  investigators,  at  least  in 
more  recent  years,  have  been  deahng,  either  in  pure  or  in  impure 
cultures,  with  the  influenza-Uke  bacillus  first  described  by  Spengler 
and  later  by  Jochmann. 

III.    PERSONAL   OBSERVATIONS. 

In  undertaking  a  study  of  the  bacteriology  of  whooping-cough, 
my  attention  was  naturally  directed  to  the  influenza-like  baciUi. 
In  the  examinations,  however,  careful  observations  were  made  upon 
other  organisms  present  with  any  degree  of  constancy. 

Technique  and  material. — The  sputum  coughed  up  during  a  paroxysm  was  obtained 
in  a  sterile  Petri  dish  and  examined  as  soon  as  possible  both  in  smear  and  culture.  As 
a  rule  the  particle  of  sputum  was  washed  in  several  successive  dishes  of  sterile  water 
to  remove  the  contaminating  mouth  organisms.  A  part  of  this  was  then  used  for 
smears  and  a  part  for  culture.  Two  smears  were  made,  one  of  which  was  stained 
with  Loeffler's  methylene  blue,  and  the  other  with  Gram's  stain.  As  a  rule  I  found 
it  useful  to  counterstain  the  latter  with  a  very  dilute  solution  of  carbolfuchsin.  In 
this  manner  the  Gram-staining  and  non-Gram-staining  organisms  could  be  very 
clearly  differentiated,  the  latter,  which  are  so  numerous  in  pertussis  sputum,  taking 
a  faint  red  stain,  while  the  former  appear  deep  blue  or  black. 

The  portion  of  sputum  used  for  culture,  after  washing,  was  introduced  into  plain 
broth  and  thoroughly  agitated;  four  tubes  of  melted  agar  at  a  temperature  of  43°  C., 
to  which  six  drops  of  defibrinated  blood  had  been  added,  were  inoculated  with  varying 
quantities  of  the  washed  sputum  and  then  plated.  In  a  few  of  the  cases  sputum  could 
not  be  obtained,  and  swabs  of  the  throat  were  used.  After  24  and  also  after  48  hours 
the  plates  were  carefully  examined  and  the  various  organisms  present  recorded. 

Pigeon-blood  agar  was  used  in  about  one-half  of  the  examinations.  In  this  it 
was  found  that  the  influenza-like  organisms,  as  a  rule,  grew  more  abundantly,  and  the 
colonies  were  larger.  However,  it  is  not  essential,  as  they  will  develop  on  any  hemo- 
globin media.  The  pigeon-blood  is  obtained  by  passing  the  needle  of  a  Luer  syringe 
into  the  heart  of  the  bird  at  a  point  just  above  the  large  breast-bone  under  the  left 
wing.  About  5  c.c.  may  be  obtained  at  one  time  from  the  heart  without  injuring 
the  pigeon.  This  blood  is  at  once  defibrinated  with  a  wire,  and  then  may  be  used 
immediately,  or  kept  in  the  ice-box  for  future  use. 

Bacteriology  of  the  cases.— The  sputum  of  6i  cases  of  pertussis 
have  been  studied  in  smear  and  culture.     In  most  cases  one  specimen 


lo  David  J.  Davis 

was  obtained,  but  in  some  cases  several  specimens  were  studied.  In 
a  number  of  the  cases  I  examined. the  sputum  in  hanging  drop, 
having  in  mind  especially  the  protozoa  reported  by  several  to  have 
been  found  in  this  way.  Nothing  was  seen  which  could  be  taken 
for  protozoa,  except  occasionally  a  ciliated  epithelial  cell.  The 
leucocytes  and  pus  cells  vary  a  great  deal  in  their  appearance,  and 
one  might  mistake  some  of  these  for  ameba.  Stained  preparations 
frequently  show  ciliated  epithelial  cells,  and  also  numerous  large, 
flat  epithelial  cells  and  polymorphonuclear  leucocytes,  but  nothing 
that  could  be  interpreted  as  protozoa  was  seen. 

A  number  of  specimens  of  whooping-cough  sputum  were  examined 
with  the  ultramicroscope  with  the  view  of  detecting  the  possible 
presence  of  an  ultramicroscopic  motile  organism.  This  was  done  by 
diluting  the  sputum  in  broth  and  then  passing  this  through  a  porcelain 
filter,  which  would  retain  all  the  microscopic  particles.  With  the 
ultramicroscope  many  small  particles  could  be  seen  in  the  filtrate 
but  they  were  all  non-motile,  and  were  probably  particles  of  proteid. 
Such  filtrates  remained  permanently  sterile  when  tested  on  various 
kinds  of  media. 

Smears  made  of  the  sputum  showed  as  a  rule  both. Gram-  and 
non- Gram-staining  bacteria.  The  Gram-staining  ones,  for  the  most 
part,  were  lanceolate  diplococci,  frequently  encapsulated.  It  is  com- 
mon to  find  these  adhering  to  the  large,  flat  epithelial  cells  so  often 
encountered  in  the  sputum.  There  is  little  doubt  that  they  belong 
to  the  pneumococcus  group.  Only  rarely  is  a  streptococcus  chain 
observed.  Occasionally  one  meets  Gram-positive  bacilli  which  are 
shown  by  culture  to  belong  usually  to  the  diphtheria  group.  Cocci 
of  the  staphylococcus  type  are  frequently  seen,  but  are  few  in  number. 
The  non- Gram- staining  organisms  are  usually  most  numerous,  and 
of  these  a  short,  small  bacillus,  practically  indistinguishable  from 
the  influenza  bacillus,  is  most  common.  Sometimes  it  exists  almost 
alone  and  in  enormous  numbers.  It  never  forms  threads  in  the 
sputum,  and  occasionally  it  is  seen  in  the  leucocytes  in  large  numbers. 
Another  non- Gram-staining  organism,  not  infrequently  met  with  in 
smear,  is  a  biscuit- shaped  diplococcus  which  appears  to  be  M. 
catarrhalis.  In  a  few  of  the  cases,  fusiform  bacilh  and  spirilla  were 
found  in  large  numbers. 


Bacteriology  of  Whooping- Cough  ii 

Upon  blood-agar  plates  the  varieties  of  bacteria  appear  so  char- 
acteristic that  after  some  experience  one  can  differentiate  them  with 
a  considerable  degree  of  certainty.  The  pneumococcus,  as  a  rule, 
has  a  typical  greenish  halo  about  the  colony,  and  the  streptococcus 
has  usually  a  wide  clear  zone  of  hemolysis.  Sometimes  diphtheria 
colonies  appear  practically  identical  with  the  streptococcus,  though 
as  a  rule  the  colony  is  whiter  and  more  elevated  and  the  zone  of 
hemolysis  not  so  wide.  Not  infrequently  the  zone  is  absent.  The 
influenza-like  organisms  form  small  transparent  delicate  blue  colonies 
without  any  zone  of  hemolysis.  Under  the  microscope,  they  -are 
nearly  homogeneous,  with  a  regular  margin.  The  colonies  of  M. 
catarrhalis  may  at  first  be  confused  with  the  influenza-like  colonies. 
There  is  no  zone  of  hemolysis,  the  colony  is  larger,  more  elevated, 
and  usually  darker  in  color.  The  margin  is  irregular  and  the  center 
is  coarsely  granular.  Some  non-hemolytic  Grampositive  diplococci, 
occasionally  met  with  in  the  first  24  hours,  may  be  mistaken  for 
influenza-like  colonies,  but  in  smears  they  are  readily  distinguishable. 

In  Table  i  is  given  a  summary  of  the  cases  of  whooping-cough 
examined,  with  data  indicating  the  more  important  organisms  found. 
Under  the  various  headings  the  approximate  number  of  organisms 
is  represented  by  +  signs. 

Of  the  61  cases  examined  there  were  33  males  and  28  females;  four  of  them  were 
under  one  year  of  age  and  three  were  adults.  The  examinations  were  made  during 
the  fall,  winter,  and  spring  of  1904-5.  At  no  time  during  this  period  was  there  an 
epidemic  of  influenza.  In  eight  of  the  cases,  bacilH  belonging  to  the  diphtheria  group 
were  found  in  the  sputum.  Most  of  these  organisms  had  granules,  and  they  usually 
produced  hemolysis  on  the  blood  plates.  In  none  of  these  eight  cases  were  they 
numerous,  and  none  gave  a  history  of  a  recent  attack  of  diphtheria,  nor  did  any  of 
them  develop  it  later.  Several  of  these  organisms  were  given  to  Dr.  Hamilton  for  further 
investigation.  In  one  case,  No.  33,  the  child  had  a  well-developed,  typical  case  of 
whooping-cough  with  no  cUnical  evidence  of  diphtheria.  A  swab  was  obtained  from 
the  throat,  which  upon  examination  showed  numerous  influenza-like  organisms,  but 
no  diphtheria  bacilli,  in  smear  or  upon  the  blood-agar  plates.  Five  days  later  the 
patient  appeared  with  a  well-developed  membrane  showing  on  examination  many 
typical  diphtheria  bacilli,  together  with  some  influenza-like  organisms. 

Under  the  streptococcus  group  are  included  the  Gram-positive 
cocci,  whose  small,  dehcate  blue  colonies  have  a  wide,  clear  zone 
of  hemolysis  about  them.  In  like  manner,  under  the  pneumococcus 
group  are  included  the  small  colonies  appearing  in  the  blood  plates, 
and  surrounded  by  a  green  zone.     While  such  a  differentiation  of 


X2 


David  J.  Davis 


TABLE    I. 
Organisms  Found  in  Pertussis  Sputum. 


No. 

Sex 

Age 

ill 

lii 

1 

3 

0  ft 
0  0 

3'-' 

1 

tococcus 
roup 

It 

Remarks 

|i 

r 

U3 

to 

Q 

I 

M 

I  yr. 

I  wk. 

+ 

0 

-f-i- 

4-4- 

4- 

Spasmodic  cough.  Distinct 
whoops  few  davs  later 

2 

F 

3yrs. 

5  wks. 

-t--t--}- 

0 

++ 

4- 

0 

3 

F 

I  yr. 

S    " 

+ 

0 

-f-i- 

4-4- 

0 

Two  examinations  made 

4 

F 

3yrs. 

13    " 

+  + 

0 

++ 

4- 

0 

Whooping-cough  followed 
diphtheria 

5 

F 

5    " 

I  wk. 

-\-  +  + 

0 

-i--f 

4- 

0 

6 

M 

3    " 

I    " 

+  +  + 

+-f- 

4- 

0 

7 

F 

4    " 

6  wks. 

0 

+4- 

+ 

4-4- 

0 

Fusiform  bacilli  and  spirilla 
present  in  large  numbers. 
Four  examinations  made 

8 

M 

3    " 

3     " 

+  +  -I- 

-f-i--i- 

++ 

+  + 

4- 

Fusiform  bacilli  and  spirilla 
numerous  in  smears 

9 

M 

8    " 

3     " 

+ 

0 

+++ 

4- 

4- 

Fusiform  bacilU  and  spirilla 
numerous 

lO 

F 

6  mo. 

4    " 

+  + 

0 

4-+ 

4- 

4- 

Five  examinations  made  before 
finding  influenza-hke  bacil- 
lus. It  appeared  during  the 
fourth  week 

II 

F 

9  yrs. 

4    " 

+  +  + 

0 

4-H- 

4- 

0 

12 

M 

2  yrs. 

5  wks. 

-I-  + 

0 

+  + 

4- 

0 

13 

F 

9    " 

IS  days 

+  + 

0 

-l--f-  + 

4- 

4- 

Three  examinations  made.  In- 
fluenza-like organisms  ap- 
peared on  15  th  day  after 
first  whoop  noted 

14 

F 

Adult 

I  wk. 

+  + 

0 

+  -i- 

4- 

0 

B.  mucosus  capsulatus  present 
in  large  numbers. 

IS 

F 

syrs. 

6  wks. 

+  -I--1- 

0 

4--}- 

4-4- 

0 

Six  examinations  made.  In- 
fluenza-like organisms  ap- 
peared during  sixth  week  of 
disease.  The  colonies  were 
slightly  hemolvtic 

i6 

F 

I  yr. 

3    " 

-H-f-J- 

0 

4-4- 

4-4- 

0 

Sputum  not  obtainable.  Cul- 
tures made  from  throat  swab 

17 

F 

syrs. 

1  wk. 

+  +  + 

0 

4-4- 

4- 

0 

Two  weeks  before  child 
whooped  examination  of 
throat  swab  was  negative 
for  influenza-Uke  organisms. 
At  this  time  she  had  a  dry 
cough 

i8 

F 

3    " 

+  + 

0 

4-4-4- 

4- 

0 

Coughed  and  vomited  but  did 
not  whoop  until  five  weeks 
after     this     examination. 
Blood  culture  negative 

10 

M 

3  !! 

S  wks. 

+  -I- 

4- 

4-4- 

4- 

0 

20 

F 

6    " 

5    " 

-f--f- 

0 

4-4-4- 

4- 

4- 

• 

21 

F 

6    " 

4    " 

-f-f 

0 

4-4- 

4- 

0 

Marked  tendency  of  influenza- 
lilce  organisms  to  form  long 
threads 

22 

F 

4    " 

3     " 

+ 

4- 

4-4- 

0 

23 

F 

3    " 

4    " 

+  + 

4-4- 

4- 

0 

24 

F 

3    " 

3     " 

-1- 

4-4- 

4- 

0 

25 

F 

2    " 

2 

+ 

4-4- 

4- 

0 

26 

M 

5    " 

10  days 

+ 

0 

4-4-4- 

0 

0 

27 

M 

7    " 

6  mos. 

+  +  + 

0 

4--h 

4- 

? 

Had  whooping-cough  in  the 
spring;  ceased  coughing 
during  summer  but  began 
again  in  the  fall.  The  ex- 
amination was  made  at  this 

28 

F 

3    " 

" 

+  + 

0 

4-4-4- 

0 

0 

time 

29 

M 

6    " 

" 

+  + 

4-4-4- 

4- 

0 

30 

F 

3    " 

Several 
days 
before 
whoop- 
ing oc- 
curred 

+ 

4-4- 

0 

0 

Throat  swab  was  obtained  for 
examination  several  days  be- 
fore whooping  occurred 

Bacteriology  of  Whooping- Cough 

TABLE  I— Continued. 


13 


No. 

Sex 

Age 

a 

$ 

i« 

a 

ll 

8  "^ 

1 

0  ^ 

1= 

fa 

r 

Remarks 

31 

M 

Adult 

iwk. 

+  + 

+ 

+  + 

0 

32 

F 

3yrs. 

2wks. 

0 

++ 

+  + 

0 

Only  one  examination  possible 

33 

M 

6    " 

4    " 

+  -I- 

0 

+  + 

+ 

A  second  examination  five 
days  after  the  first  showed  a 
typical  diphtheritic  mem- 
brane with  numerous  diph- 
theria bacilli 

34 

M 

5    " 

4  '! 

0 

0 

+++ 

+ 

0 

Only  one  examination  possible 

35 

M 

4 

3 

+  +  + 

+  + 

+ 

0 

36 

M 

4    " 

4    " 

+  +  + 

0 

+++ 

+ 

0 

37 

M 

4    " 

3     " 

+  +  + 

+.+ 

+ 

0 

38 

F 

4    " 

4    " 

+  + 

0 

++ 

+ 

0 

Obtained  pure  culture  of  in- 
fluenza-like organism  by 
having  child  cough  against 
plate  10  inches  away 

39 

M 

5    " 

2     " 

+- 

-++ 

— 

++ 

+ 

0 

40 

M 

4    " 

3     " 

— 

-++ 

0 

++ 

+ 

4- 

41 

M 

6    " 

— 

--!-+ 

0 

++ 

+ 

0 

Coughed    severely,    but    first 

heard  to  whoop  two  days 

after  examination 

42 

M 

7yrs. 

3  wks. 

++++ 

0 

+ 

+ 

0 

Almost  piu-e  cultpe  of  influ- 
enza-like organispis 

43 

F 

10    " 

Several 
weeks 

+  + 

0 

++ 

+ 

0 

44 

M 

8    •' 

" 

+  + 

0 

++ 

+ 

0 

45 

M 

g    " 

4  wks. 

+  4- 

0 

+  + 

+ 

0 

46 

M 

8  mos. 

5    " 

+++  + 

0 

+ 

0 

0 

Nearly  pure  growth  of  influ- 
enza-like organism 

47 

M 

7   " 

5    " 

+4-++ 

0 

++  + 

4- 

0 

48 

M 

syrs. 

5    " 

+ 

0 

+  +  + 

+ 

0 

Influenza-like  bacilli  show 
marked  tendency  to  thread 
formation  and  phleomor- 
phism 

49 

M 

3    " 

4    " 

0 

+  +  + 

+ 

+ 

0 

Only  one  specimen  obtainable 

50 

M 

6    " 

6    " 

+ 

0 

+++ 

+ 

0 

Nearly  recovered  when  exami- 
nation was  made 

SI 

M 

7    " 

5     " 

+  + 

? 

+  + 

4- 

0 

52 

F 

4    " 

4    " 

4- 

+ 

++ 

4-  + 

0 

Complicated  with  syphiUs 
UTiooping-cough    followed 

53 

M 

17    • 

4     " 

+  +  + 

0 

+++ 

.  +  + 

0 

pneumoma 

54 

M 

5     " 

6    " 

+ 

? 

+++ 

+ 

0 

Very  severe  vomiting 

55 

F 

6    " 

6    " 

+ 

+  + 

+  +  + 

4- 

0 

Whooping-cough    followed 
pneumonia  and  empyema 

56 

M 

4    " 

3     " 

+  +  + 

0 

++++ 

0 

0 

57 

M 

10     " 

5     " 

++++ 

0 

+ 

0 

0 

Examination  by  throat-swabs 

S8 

F 

8  mos. 

3     " 

+  + 

0 

+  + 

+ 

0 

59 

F 

3  vrs. 

2     " 

+  +  + 

0 

+  + 

+  + 

0 

60 

M 

2    " 

3  days 

+ 

0 

+++ 

4-  + 

0 

Blood  culture  was  negative 

61 

M 

2    " 

3     " 

+ 

+  + 

+++ 

+ 

+ 

pneumococcus  and  streptococcus  may  not  be  absolutely  correct,  for 
practical  purposes  it  probably  gives  an  approximately  accurate  indi- 
cation of  the  occurrence  of  these  organisms.  As  v^^ill  be  seen  from 
the  table,  both  of  them  are  found  in  practically  every  case.  Strep- 
tococci are,  as  a  rule,  not  nearly  so  numerous  as  pneumococci.* 

*  In  the  pneumococcus  group  is  included  SchottmuUer's  Streptococcus  tnitior  seu  viridans 
inasmuch  as  it  produces  green  colonies  on  blood-agar  plates.  Inulin  tests  of  the  various  colonies  were 
not  made.     Since  these  green   colonies  were  found  constantly  in  the  throats  of  all  cases,  not  only  of 


14  David  J.  Davis 

Organisms  corresponding  to  M.  catarr halts,  as  described  by  Pfeif- 
fer,  occurred  in  large  numbers  in  a  few  cases.  They  occur  in  pairs, 
being  indistinguishable  in  smear  from  the  meningococcus.  Usually, 
when  present,  they  are  seen  in  large  numbers  inside  the  leucocytes 
in  the  sputum. 

Influenza-like  organisms. — In  these  cases  attention  was  particularly 
directed  to  the  occurrence  of  the  influenza-like  bacilli.  Usually  they 
were  found  in  the  first  examination.  In  this  series  44  of  the  61  cases 
required  but  one  examination 'to  find  such  bacilli.  In  12  of  the  56 
cases  in  which  they  were  found,  two  or  more  examinations  were 
necessary  to  find  them.  In  five  of  the  61  cases  they  were  not  found, 
but  in  three  of  these  only  one  examination  was  possible;  in  the 
remaining  two,  two  examinations  were  made  in  one,  and  four  in 
the  other,  with  negative  results.  In  two  cases  the  organisms  were 
found  several  days  before  the  patients  had  been  heard  to  whoop, 
but  they  had  at  this  time  a  severe  cough  and  expectorated  considerable 
mucus.  In  six  cases  where  the  children  were  known  to  have  been 
exposed  to  the  disease,  and  were  showing  some  symptoms  of  a  dry 
cough,  swabs  of  the  throat  were  examined  for  these  bacilli,  but  in 
none  were  they  found.  They  all  developed  typical  whooping-cough 
in  from  one  to  two  weeks  later.  As  to  the  length  of  time  these  organ- 
isms persist  in  the  throat,  present  data  will  not  suffice.  One  case 
that  had  developed  pertussis  in  the  spring  and  had  ceased  coughing 
during  the  summer,  began  to  whoop  again  in  the  fall,  i.  e.,  about  six 
months  from  the  first  attack.  The  sputum  at  this  time  contained 
an  abundance  of  the  bacilli.  A  brother  of  this  child,  who  also  had 
pertussis  in  the  spring  and  recovered  during  the  summer,  but  who 
did  not  cough  in  the  fall,  also  showed  large  numbers  of  the  same 
bacilli  in  his  throat  at  this  time.  The  organisms  were  as  abundant 
in  the  adults  as  in  the  young  children.  It  may  therefore  be  said  that 
these  bacilli  occur  practically  constantly  in  the  throats  during  the 
spasmodic  period,  and  even  slightly  before  this,  but  probably  not 
during  the  initial  stage  of  the  disease. 

The  bacilli  were  often  extremely  numerous,  in  a  few  cases  being 
nearly  in   pure   culture;    this,    however,    depends   largely   on   how 

whooping-cough,  but  of  the  diseases  given  in  Table  2,  as  well  as  in  the  20  normal  throats  examined, 
they  should  be  considered  the  normal  inhabitants  of  the  throat.  It  is  of  interest  that  organisms  of  this 
type  are  practically  absent  on  the  normal  nasal  mucosa  as  shown  by  the  examination  of  the  same  20 
normal  cases  that  yielded  them  in  abundance  from  the  pharyngeal  mucosa. 


Bacteriology  of  Whooping- Cough  15 

thoroughly  the  sputum  is  washed.  In  smears  of  the  sputum  they 
sometimes  appear  as  the  only  organism  present.  In  a  few  cases 
the  leucocytes  in  the  sputum  were  crowded  with  them,  but  generally 
they  were  outside  the  cells. 

Morphology. — The  organism  is  a  small,  short,  non-motile  bacillus, 
which  does  not  take  Gram's  stain,  does  not  have  a  capsule,  and  does 
not  form  spores.  It  stains  more  deeply  at  the  ends,  especially  with 
methylene  blue,  and  therefore  may  be  mistaken  for  a  small  diplo- 
coccus.  It  stains  especially  well  with  dilute  carbolfuchsin.  In 
sputum  the  bacillus  is  nearly  always  single,  but  occasionally  two  or 
three  bacilH  may  be  seen  in  a  chain.  Their  morphological  char- 
acteristics in  sputum  are  very  uniform.  In  the  culture,  however, 
it  is  quite  different.  In  many  of  the  strains  there  is  a  marked  tendency 
to  chain  and  thread  formation.  Frequently  the  organisms  occur  as 
coccus  forms  and  in  thick,  curved,  or  S-shaped  threads.  Sometimes 
threads  may  be  seen  reaching  across  the  field  of  the  microscope,  and 
much  thicker  than  the  single  bacillus.  As  was  stated,  they  are  never 
seen  in  smears  direct  from  the  throat,  but  may  occur  in  the  first 
generation  in  the  blood  plates,  or  develop  later.  They  are  usually 
more  marked  in  a  culture  a  few  days  old.  When  one  meets  these 
for  the  first  time  he  is  convinced  that  he  has  a  mixed  culture  or  some 
entirely  different  organism.  These  peculiar  irregular  forms  agree 
well  with  Pfeiffer's  pseudo-influenza  organisms,  which  he  isolated 
from  cases  of  broncho-pneumonia.  However,  this  fact  should  be 
emphasized,  that  many  of  the  strains  do  not  show  this  tendency  to 
any  extent  under  any  circumstances,  and,  so  far  as  this  point  is  con- 
cerned, agree  with  Pfeiffer's  description  of  the  real  influenza  bacillus. 
As  regards  the  size  of  the  bacillus,  the  various  strains  all  appear  about 
the  same  in  sputum,  and,  compared  with  the  few  influenza  bacilli 
that  I  have  been  able  to  obtain,  show  practically  no  differences. 
In  cultures,  however,  bacilli  of  various  strains  from  whooping-cough 
vary  in  size  so  much  among  themselves  that  a  comparison  is  difficult. 
Certainly  many  of  the  whooping-cough  strains  are  as  small  as  organ- 
isms from  typical  clinical  cases  of  influenza. 

Cultural  characteristics. —  The  organisms  from  pertussis  corre- 
spond in  their  cultural  characteristics  in  every  way  to  Pfeiffer's  bacillus. 
It  is  a  strict  aerobe.     On  plates  the  colonies  are  small,  moist  and 


1 6  David  J.  Davis 

dew-drop-like.  They  have  a  dehcate  blue  color  and  do  not  hemolyze. 
In  one  case  (No.  15)  only,  did  I  observe  a  very  narrow  but  distinct 
clear  zone  of  hemolysis  about  the  colonies.  This  persisted  through 
two  generations,  and  then  disappeared  and  could  not  be  obtained 
again.  The  colonies,  if  not  very  numerous,  especially  on  pigeon 
blood,  will  sometimes  grow  very  large.  Some  have  been  observed 
over  2  mm.  in  diameter.  As  a  rule  they  are  J  mm.  or  less  in 
diameter,  and  not  infrequently,  if  numerous  on  the  plate,  require 
the  aid  of  a  hand  lens  to  be  seen.  They  do  not  increase  in  size  after 
from  24  to  36  hours.  Upon  blood-agar  slants  they  produce  a  delicate 
growth  and  the  colonies  remain  discrete. 

In  every  case,  after  getting  the  organism  in  pure  culture  on  blood 
media,  it  was  tested  on  several  kinds  of  non-hemoglobin  media, 
usually  Loeffler's  blood  serum,  plain  agar,  and  milk.  Many  of  the 
strains  were  tested  on  serum  agar,  serum  broth,  ascites  agar,  and 
various  kinds  of  sugar  media.  In  none  of  these  did  any  of  the  organ- 
isms develop.  Particular  attention  should  be  called  to  the  fact  that 
there  is  not  infrequently  in  human  serous  fluids  a  small  amount  of 
hemoglobin,  and  if  such  fluids  are  used  growth  will  occur.  This 
fact  may  explain  some  of  the  reports  in  the  Hterature,  such  as  that 
of  Elmassian,  of  organisms  from  pertussis  cases  growing  upon  serum 
media.  To  be  sure  of  proper  results,  one  should  examine  such  fluids 
with  a  spectroscope  before  using. 

Media  to  which  yolk  of  egg  was  added  did  iiot  yield  any  growth. 
Bile  was  added  to  agar,  and  some  growth  was  obtained  with  this  in 
the  first  transplantation,  but  continuous  growth  did  not  occur.  The 
organism  in  piire  culture  did  not  grow  on  hematin  agar.  Upon  CO- 
hemoglobin  they  grow  as  well  as  upon  ordinary  oxyhemoglobin. 
Inasmuch  as  oxygen  is  necessary  for  their  growth,  the  CO- hemoglobin 
made  by  passing  a  stream  of  CO  through  blood  for  half  an  hour,  was 
necessarily  exposed  to  the  air,  and  a  small  amount  was  consequently 
changed  back  to  oxyhemoglobin,  which  might  explain  this  growth. 
The  abundant  growth  suggests,  however,  that  the  CO-hemoglobin 
may  also  be  available,  though  it  is  perhaps  impossible  to  prove  this. 
Growth  is  obtained  in  Hquid  media  to  which  a  drop  or  two  of  blood 
is  added.  Plain  broth  plus  a  small  amount  of  pigeon's  blood  is  a 
good  medium.     The  blood  corpuscles  soon  sink  to  the  bottom,  leaving 


Bacteriology  of  Whooping- Cough  17 

a  perfectly  clear  fluid  above.  When  inoculated,  this  becomes  turbid 
as  a  result  of  the  growth,  such  a  culture  being  very  convenient  for 
inoculations.  The  bacilli  grow  in  milk,  and  also  in  the  various  kinds 
of  sugar  media,  if  a  little  hemoglobin  is  added,  but  no  change  occurs 
in  any  of  them. 

Only  a  very  small  amount  of  hemoglobin  is  required  for  the  growth 
of  these  bacilH.  One  may  dilute  blood  with  sterile  water  many 
times,  and  if  a  few  drops  of  this  laked  solution  is  added  to  agar  a 
clear  transparent  medium,  scarcely  colored  at  all,  is  obtained,  upon 
which  the  bacilU  grow  in  abundance.  This  is  a  very  convenient 
medium  to  use  for  their  culture.  The  organism  is  rather  dehcate 
and  must  be  transferred  every  five  days  to  make  sure  of  growth. 
They  seem  to  live  somewhat  longer  in  liquid  than  on  soHd  media. 
They  are  very  susceptible  to  drying,  and  a  temperature  of  42°  C.  for 
a  few  hours  is  fatal.  The  bacilU  have  been  observed  through  many 
successive  generations  without  undergoing  any  essential  change. 
Some  strains  have  been,  transplanted  every  four  or  five  days  for  six 
months,  and  their  morphology  and  cultural  characteristics  remained 
unaltered. 

These  bacilli  are  thrown  out  into  the  air  in  large  numbers  during 
the  coughing  spell  of  the  child,  and  if  one  is  in  front  of  the  patient 
he  is  sure  to  inhale  some  of  them.  In  five  cases  blood-agar  plates 
were  held  in  front  of  the  patient  from  6  to  12  inches  eway  during  the 
spasm,  and  in  every  instance  the  baciUi  were  isolated.  There  are 
always  other  organisms  on  the  plate,  and  usually  the  colonies  are 
not  pure,  for  the  bacilH  are  carried  out  from  the  throat  in  particles 
of  spray,  which  one  would  expect  to  contain  more  than  one  kind  of 
bacteria.  With  babies,  where  it  is  difficult  or  impossible  to  obtain 
sputum,  examination  for  the  bacilU  may  be  made  in  this  way. 

Symbiosis. — Grassberger^  has  called  particular  attention  to  the 
occurrence  of  very  large  influenza  colonies  in  the  immediate  neigh- 
borhood of  colonies  of  other  bacteria,  when  grown  with  them 
on  blood-agar  plates.  He  worked  especially  with  staphylococcus, 
but  the  same  effect  is  obtained  when  influenza  bacilli  are  grown  with 
many  other  varieties.  He  observed  influenza  colonies  as  large  as 
4  mm.  in  diameter,  grown  in  this  manner,  which  in  pure  culture 
are  usually  only  ^  mm.  or  less.     Jochmann  also  observed  this  same 

•  Ztschr.  /.  Hyg.,  7*97,  25,  p.  453- 


i8  DAvm  J.  Davis 

phenomenon  in  connection  with  his  B.  pertussis  Eppendorf.  It  is  well 
known  that  influenza  bacilli  will  live  and  develop  through  several 
generations  on  non-hemoglobin  media,  when  grown  in  mixed  culture. 
Pfeififer,  before  he  discovered  the  use  of  hemoglobin  for  influenza 
culture,  noted  the  growth  of  influenza  bacilli  on  agar  smeared  with 
sputum,  but  could  not  successfully  transplant  them  on  this  medium 
in  pure  culture.  M.  Neisser'  obtained  mixed  cultures  of  B.  xerosis 
and  B.  influenzae  from  conjunctivitis  in  measles,  and  grew  them 
through  20  generations  on  plain  agar.  Strains  of  hemophilous  bacilli 
from  throats  of  whooping-cough,  measles,  and  scarlet  fever  were 
in  like  manner  carried  through  many  generations  in  mixed  culture. 
With  dead  B.  xerosis  or  its  extracts,  no  growth  occurred.  He  says 
the  presence  of  a  living  organism  is  necessary  to  produce  this  sym- 
biotic effect. 

Various  attempts  have  been  made  to  explain  the  mechanism  of 
symbiosis.  Ghon  and  Preyss^  think  it  may  be  due  to  a  reduction 
of  the  hemoglobin  or  hematin  by  some  substance  produced  by  the 
foreign  organisms,  thereby  rendering  the  iron  more  available.  Luers- 
sen3  found  the  favorable  substance  residing  in  the  cell  body  of  the 
bacteria,  and  not  in  any  product.  He  also  claimed  that  filtrates, 
made  after  several  days'  growth,  were  favorable  for  the  development 
of  the  influenza  bacillus. 

Frequently,  in  the  blood-agar  plates  inoculated  from  the  whooping- 
cough  sputum,  there  was  observed  a  conspicuous  cluster  of  influenza- 
like colonies  surrounding  other  colonies  present  on  the  plates,  such 
as  those  of  streptococcus,  staphylococcus,  and  pneumococcus.  They 
were,  in  this  location,  not  only  larger,  but  apparently  more  numerous, 
than  when  farther  away  from  the  foreign  colonies.  In  order  to 
obtain  such  an  arrangement,  it  seems  necessary  to  have  a  large 
number  of  influenza  colonies  in  the  plate,  and  a  few  of  the  foreign 
organisms.  When  the  former  colonies  are  numerous,  they  are  often 
so  small  that  a  hand  lens  is  necessary  to  see  them;  but  around  the 
foreign  colonies,  as  a  result  of  some  favorable  influence,  they  become 
much  larger  and  are  more  easily  visible.  This  appearance  may 
be  easily  obtained  by  sowing  a  blood-agar  plate  densely  with  the 

'  Deutsche  med.y  Wchnschr.,  IQ03,  29,  p.  462.  '  Ibid.,  p.  434. 

'  Centralbl.  f.  BakL,  Abt.  I,  Orig.  1904,  35,  p.  531. 


■   "'^'VEV^; 


Bacteriology  or  Whooping- Cough  19 

pertussis  bacillus,  and  sparsely  with  some  other  organism,  such  as 
staphylococcus.  In  24  hours  the  symbiotic  relationship  is  usually 
evident. 

If  pertussis  sputum  containing  large  numbers  of  the  influenza-like 
bacilli,  as  it  commonly  does,  is  spread  on  plain  agar  or  Loeffler's 
blood-serum,  these  bacilH  will  often  grow  in  abundance.  Other 
germs  are  always  present  under  these  circumstances,  and  undoubtedly 
their  growth  has  much  to  do  in  causing  the  influenza-like  bacilU  to 
develop  on  the  hemoglobin-free  media;  for,  in  sterile  sputum  alone, 
the  latter  do  not  grow.  If  transferred  thus  in  impure  culture  upon 
plain  media,  they  often  survive  several  generations,  and  their  growth 
may  be  easily  determined  by  making  blood-agar  plates  at  the  time 
of  each  transplantation.  It  was  practically  this  technique  that 
Czaplewski  and  Hensel,  and  others  who  obtained  similar  results 
later,  used  to  isolate  their  bacillus,  and  one  is  strongly  tempted  to 
believe  that  they  have  thus  been  cultivating  in  impure  culture  the 
same  hemophilous  bacillus  found  by  Spengler  and  Jochmann.  As 
already  stated,  this  was  Spengler's  explanation  of  their  results.  Such 
an  interpretation  seems  far  more  reasonable  than  the  possibiHty  that 
Czaplewski 's  organism,  found  as  frequently  as  he  reported,  was  an 
entirely  different  organism  from  the  hemophilous  bacillus  found  by 
so  many. 

The  growth  of  the  several  strains  of  influenza-like  bacilH  from 
pertussis  was  tested  upon  various  media,  to  which  were  added  dead 
bacteria  (staphylococci,  streptococci)  and  filtrates  of  cultures;  the 
results  were  uniformly  negative.  All  of  the  experiments  performed 
indicated  that  the  symbiosis  is  dependent  on  the  presence  of  the 
Hving  organisms. 

The  symbiotic  relation  of  other  bacteria  to  the  influenza-like 
organisms  is  shown  by  the  increased  pathogenicity  of  the  latter  for 
animals.  This  will  be  more  fully  discussed  under  the  head  of  animal 
experiments. 

IV.    ANIMAL  EXPERIMENTS. 

Literature. — The  results  obtained  by  the  inoculation  of  whooping- 
cough  virus  into  animals  by  various  investigators  have  for  the  most 
part  been  negative.  The  older  reports  of  producing  the  disease  in 
dogs  can  hardly  be  considered  seriously.     Afanassiew  reported  that 


20  David  J.  Davis 

he  produced  a  real  whooping-cough  in  animals  by  the  inoculation 
of  his  organism,  but  these  results  have  never  been  substantiated. 
Ritter's  animal  experiments  were  not  conclusive.  Injected  into  the 
circulation,  his  diplococcus  produced  no  effect.  Czaplewski  and 
Hensel,  and  also  Zusch,  obtained  no  results  in  animals  with  their 
organism.  Koplik  reported  that  intravenous  inoculation  with  his 
bacillus  produced  suppurative  arthritis,  that  5  c.c.  of  culture  inocu- 
lated into  white  mice  killed  in  one  week,  and  that  the  organisms  were 
present  in  the  blood;  but  that  inoculation  of  guinea-pigs  and  rabbits 
with  sputum  gave  negative  results.  Vincenzi's  results  were  negative. 
Elmassian,  with  his  organism,  produced  death  in  young  guinea-pigs 
by  intraperitoneal  inoculation  in  24  hours  or  less,  and  isolated  the 
bacillus  from  the  fluids  and  heart's  blood;  there  was  marked  pha- 
gocytosis. Intravenous  injection  into  rabbits  produced  some  cache- 
xia, while  pigeons  and  mice  did  not  react.  Manicatide  obtained  no 
results  by  inoculating  his  bacillus  ''z"  into  the  nose  and  trachea  of 
guinea-pigs,  cats,  dogs,  and  monkeys.  Jochmann  and  Krause  were 
not  able  to  produce  any  effect  either  with  sputum  or  pure  cultures  of 
their  bacillus,  when  inoculated  into  the  nasal  cavities,  throat,  or 
peritoneum  of  animals.  On  the  other  hand,  Smit  found  Jochmann's 
bacillus  pathogenic  for  young  guinea-pigs  by  peritoneal  injections, 
and  that  it  also  produced  lameness,  general  infection,  and  death  in 
rabbits  by  intravenous  injection.  He  also  produced  double-sided 
conjunctivitis  in  a  rabbit,  and  obtained  the  bacillus  in  the  exudate. 
A  distinct  reaction  occurred  in  the  horse  when  injections  were  made 
with  the  bacillus  for  immunizing  purposes.  Wollstein  reports  nega- 
tive results  with  the  influenza-like  organism  from  intraperitoneal 
and  subcutaneous  inoculations  in  white  mice,  guinea-pigs,  and  rabbits, 
and  also  from  a  subdural  inoculation  in  a  rabbit. 

For  comparison  with  the  above  results,  we  may  briefly  refer  to 
results  obtained  with  Pfeiffer's  bacillus.  Inoculation  experiments 
with  the  sputum  of  influenza  patients  and  with  pure  cultures  of  the 
bacillus  upon  guinea-pigs,  rats,  mice,  and  doves  are  generally  without 
result.  In  rabbits  and  monkeys  some  symptoms  have  been  produced. 
Pfeiffer,  by  injecting  the  bacilli  into  the  lungs  of  monkeys,  observed 
symptoms  simulating  those  of  influenza,  and  by  intratracheal  injec- 
tion of  the  bacilli  killed  a  monkey  in  eight  hours.     There  being  no 


Bacteriology  of  Whooping- Cough  21 

anatomical  change  visible,  this  latter  result  was  attributed  to  the 
toxic  products — surely  a  most  doubtful  conclusion.  In  rabbits,  by 
intravenous  inoculation  of  living  baciUi,  dyspnea  and  marked  weak- 
ness were  produced,  which  Pfeiffer  also  attributes  to  toxins,  because 
the  same  result  was  produced  with  dead  bacilli.  Kolle  and  Delius 
substantiated  these  results,  and  also  showed  that  influenza  bacilli 
would  develop  in  the  peritoneal  cavity  of  animals,  especially  guinea- 
pigs.  The  exudate  contained  many  bacilli,  partly  free  and  partly 
in  the  cells.  They  were  able  to  raise  the  virulence  of  the  bacilli  by 
successive  animal  inoculations,  and  in  filtrates  of  cultures  obtained 
a  specific  but  very  labile  poison.  Jacobson^  increased  the  virulence 
of  the  influenza  organism  by  simultaneous  injection  with  streptococci, 
and  Slatineano^  raised  the  virulence  by  injecting  lactic  acid  at  first 
and  one-half  hour  later  the  influenza  bacilli. 

Experiments  on  lower  animals. — The  influenza-like  bacilli  isolated 
from  the  whooping-cough  patients  in  this  series  showed  on  the  whole 
a  low  degree  of  virulence  for  animals;  however,  some  very  definite 
results  were  obtained.  Guinea-pigs  were  found  most  satisfactory  for 
this  purpose.  White  rats  are  susceptible,  but  less  so  than  guinea-pigs; 
rabbits,  especially  young  ones,  are  also  susceptible  when  inoculated 
intraperitoneally.  But  few  inoculations  were  made  in  these  animals, 
however.  Two  blood-agar  cultures  inoculated  intraperitoneally  into 
guinea-pigs  will  rarely  kill  in  24  hours.  Usually  the  animal  is  sick  the 
next  day  after  inoculation,  but  will  gradually  recover.  If  the  amount 
is  increased,  death  will  usually  occur  in  24  hours  or  less,  and  from  the 
peritoneal  cavity,  pleural  cavity,  and  heart's  blood,  the  bacilli  are 
obtained  in  abundance.  In  the  blood  the  bacilli  are  so  numerous  at 
times  that  they  may  be  found  without  great  difficulty  in  smear.  A 
few  drops,  allowed  to  run  over  the  surface  of  an  agar  slant,  usually 
shows  an  abundance  of  colonies  in  24  hours.  Blood  removed  from 
the  heart  before  death  also  shows  the  presence  of  baciUi,  so  that  their 
occurrence  is  not  due  to  a  postmortem  invasion,  but  there  seems  to 
be  an  actual  multipUcation  of  them  in  the  living  blood. 

When  the  animal  dies  in  24  hours  or  thereabouts  after  inoculation 
into  the  peritoneal  cavity,  the  peritoneal  and  pleural  exudates  are 
clear,  serous  in  character,  and  soon  clot  on  standing.     The  bacilli 

•  Archiv  de  mid.  expir.,  1901,  13,  p.  425. 

*  Comp.rend.delaSoc.de  Biol.,  igoi,  s^yV-^So 


22  David  J.  Davis 

are  very  numerous.  But  few  polynuclear  leucocytes  are  present,  and 
these  are  usually  filled  with  the  bacteria.  If  the  exudate  of  an  animal 
inoculated  with  an  amount  insufficient  to  kill  in  24  hours  is  examined 
about  the  second  or  third  day,  a  very  different  picture  is  presented. 
The  exudate  is  purulent  and  contains  flakes  of  fibrin.  The  polynu- 
clear leucocytes  are  very  abundant,  and  are  busily  engaged  ingesting 
the  bacteria.  There  are  also  present,  in  less  numbers,  large  mononu- 
clear cells.  These  cells  often  contain  one  or  more  polynuclear 
leucocytes  filled  with  bacilh.  The  ingested  leucocytes  may  be  seen 
in  various  stages  of  intracellular  digestion,  some  being  nearly  perfect, 
while  others  show  nothing  but  fragments  of  the  nucleus. 

A  number  of  animal  experiments  were  made  to  determine  the 
effect  of  introducing  another  organism  with  the  influenza-like  bacilh 
into  the  peritoneal  cavity.  This  had  been  done  with  the  influenza 
bacillus  and  the  streptococcus  by  Jacobson,  who  found  that  the 
virulence  of  the  bacilli  could  thus  be  increased  so  that  much  smaller 
doses  of  the  bacilh  alone  would  be  sufficient  to  kill.  He  obtained 
both  the  bacilh  and  streptococci  in  the  body  fluids  and  heart's  blood. 
In  my  experiments  a  non-virulent  Staphylococcus  pyogenes  aureus 
was  chiefly  used.  Two  c.c.  of  a  broth  culture  of  this  organism  pro- 
duced no  effect  when  inoculated  into  the  peritoneal  cavity  of  guinea- 
pigs.  When  J  c.c.  of  this  culture  was  inoculated  with  two  blood- 
agar  slants  of  the  pertussis  organism,  which,  as  above  stated,  rarely 
killed,  death  invariably  followed  in  24  hours  or  less.  From  the 
peritoneal  exudate  both  organisms  were  isolated;  from  the  heart's 
blood,  however,  as  a  rule  only  the  influenza-Hke  bacilli  were  obtained, 
and  no  staphylococci.  In  two  animals,  where  large  amounts  of 
staphylococci  were  injected,  a  few  were  obtained  also  in  the  heart's 
blood.  After  passing  the  bacilli  in  symbiosis  with  staphylococci 
through  six  animals,  one  blood- agar  slant  alone  would  kill  an  animal 
in  24  hours,  i.  e.,  its  virulence  had  been  more  than  doubled.  The 
effect  of  M  catarrhalis  and  an  avirulent  streptococcus  upon  the 
influenza-like  bacilli  was  found  to  be  very  similar  to  that  of 
staphylococcus,  i.  e.,  the  bacilli  were  distinctly  more  virulent  when 
associated  with  these  organisms  than  when  inoculated  alone.  In  all 
these  experiments  controls  were  made  by  inoculating  animals  with 
the  same  or  larger  amounts  of  the  organisms  separately. 


Bacteriology  of  Whooping- Cough  23 

The  increased  pathogenicity  of  this  organism  when  growing  with 
another  organism  is  of  great  importance,  and  deserves  much  more 
attention  and  study  than  has  been  given  to  it.  This  property  is 
directly  associated  with  the  other  symbiotic  phenomena  noted  earher 
in  the  paper,  and  probably  depends  upon  the  same  factors.  Under 
the  conditions  in  which  we  find  these  bacilli  in  the  throat,  for  instance, 
they  are  always  associated  with  other  organisms,  and  therefore  are 
thriving  under  circumstances  which  permit  a  manifestation  of  their 
greatest  virulence  and  most  luxuriant  growth.  It  is  also  quite  pos- 
sible that  the  injurious  products  of  such  growth  may  be  not  only 
more  abundant,  but  even  of  a  different  and  more  toxic  character. 
It  would  therefore  be  improper  to  draw  conclusions  from  data 
obtained  by  growing  the  organisms  in  pure  culture. 

If  one  injects  intraperitoneally  two  or  three  drops  of  lactic  acid  in 
2  c.c.  of  water  one-half  hour  before  the  injection  of  influenza-Hke 
bacilli  from  pertussis,  the  resistance  of  the  animal  toward  them  is 
diminished,  and  it  will  therefore  succumb  to  smaller  amounts  of 
bacilli  than  otherwise.  This  is  due,  in  all  probability,  to  the  effect 
of  the  acid  upon  the  leucocytes,  rendering  them  unable  successfully 
to  combat  the  bacilli.  This  corresponds  with  the  results  obtained 
by  Slatineano,  who  used  this  method  to  raise  the  virulence  of  influ- 
enza bacilH. 

Sputum  from  whooping-cough  patients,  containing  large  numbers 
of  the  bacilli,  and  also  pure  cultures  of  this  organism,  were  inoculated 
into  the  throat  and  nasal  cavities  of  a  monkey,  with  no  effect. 
Another  monkey,  inoculated  in  the  same  way  with  influenza  bacilli 
from  a  typical  case  of  acute  influenza,  hkewise  showed  no  reaction. 

The  reported  attempts  to  produce  whooping-cough  in  human 
beings  by  inoculations  are  of  Httle  significance.  After  producing 
pertussis-Hke  symptoms  in  a  rabbit  with  a  fungus  isolated  from 
pertussis  sputum,  decaying  oranges,  apples,  etc.,  which  he  considered 
the  cause  of  the  disease,  Tschamer^  and  his  assistant  inhaled  some 
of  the  pulverized  mold.  In  eight  days  they  had  symptoms  of  spas- 
modic coughing,  and  the  fungus  was  found  in  the  sputum.  In  this 
manner  the  etiology  was  settled.  Ritter^  and  his  assistants  inhaled 
cultures  of  his  diplococcus  with  no  result  other  than  a  slight  mechani- 

•  Jahrbuch  f.  Kinderh.,  1876,  10,  p.  174.  »  Berl.  klin.  Wchnschr.,  1893,  30,  p.  1154- 


24  David  J.  Davis 

cal  irritation.  Czaplewski  states  that,  while  working  with  his 
organism,  he  was  taken  with  a  coryza  accompanied  with  some  cough, 
which  lasted  for  about  a  week.  He  obtained  his  bacillus  from  his 
nose  and  throat  in  large  numbers. 

Human  inoculation. — As  no  attempts  are  on  record  to  inoculate 
man  directly  with  the  influenza-like  bacillus  from  pertussis,  it  seemed 
desirable  to  make  such  a  test  with  the  view  of  determining  its  pos- 
sible pathogenicity.  A  young  man,  who,  according  to  his  statement 
and  also  that  of  his  relatives,  had  never  had  whooping-cough,  agreed 
to  submit  to  inoculation  with  hemophilous  organisms  isolated  from 
the  pertussis  cases.  This  was  done  with  his  full  consent,  and  he  was 
informed  concerning  the  character  of  the  material  inoculated  and  the 
possible  results  anticipated.  He  was  healthy  in  every  way,  and  an 
examination  of  his  throat  made  previous  to  the  inoculation  showed 
only  the  usual  bacterial  flora  of  the  normal  throat.  There  were  no 
influenza-like  organisms  present.  Pure  blood-agar  cultures,  24  hours 
old,  of  influenza-Hke  bacilli,  isolated  from  an  uncompKcated  case  of 
whooping-cough,  were  smeared  upon  the  nasal  mucosa  and  the  ton- 
sils. On  the  second  day,  almost  exactly  48  hours  after  the  inocula- 
tion, the  patient  complained  of  a  chilly  sensation,  a  cold  sweat,  some 
headache  and  weakness.  His  temperature  rose  from  normal 
(98.4°  F.  the  day  previous)  to  100.2°  F.  The  same  evening  it 
registered  100. i°F.  The  next  morning  it  was  normal  again,  but 
rose  during  the  day  to  99.5°.  The  third  day  his  temperature  was 
practically  normal  and  continued  so  from  that  time  on.  He  com- 
plained somewhat  of  headache  and  of  feeling  unwell,  and  on  the 
second  morning  said  he  coughed  a  little.  His  throat,  upon  examina- 
tion when  the  first  symptoms  appeared,  was  shghtly  hyperemic;  the 
next  morning  there  was  present  a  thick  layer  of  mucus  upon  the 
pharyngeal  wall,  and  from  this  time  on  he  coughed,  or  rather  hacked 
up  large  quantities  of  a  stringy,  tenacious  mucus.  This  condition 
continued,  becoming  gradually  less  marked,  and  was  still  apparent 
at  the  end  of  four  weeks.  After  the  first  few  days  he  felt  perfectly 
well,  complaining  only  of  slight  discomfort  in  the  throat  and  the  light 
cough.  This  cough  was  not  spasmodic  and  did  not  resemble 
whooping-cough.     The  leucocyte  count  on  the  second  day  was  9,200. 

Bacteriological  examination  of  the  mucus  from  the  throat,  obtained 


Bacteriology  of  Whooping- Cough  25 

on  the  first  day  of  symptoms  revealed  almost  a  pure  culture  of  the 
influenza-like  bacilli.  They  were  present  both  in  the  throat  and  on 
the  nasal  mucosa,  in  enormous  numbers.  Direct  smears  of  the 
expectorated  mucus  showed  practically  no  other  organism.  They 
were  identical  in  every  detail  with  the  organisms  inoculated.  Exami- 
nations of  the  throat  were  made  every  few  days  for  four  weeks,  after 
which  time  the  patient  was  not  accessible.  The  number  of  organisms 
present  became  gradually  less,  but  at  the  end  of  that  time  they  were 
still  present  in  considerable  numbers. 

From  this  experiment  we  must  conclude  that  this  organism 
develops  in  the  human  throat  when  implanted  there,  and  is  capable 
of  causing  a  distinct  reaction.  The  fact  that  pertussis  was  not  pro- 
duced by  this  one  inoculation  by  no  means  excludes  this  organism 
as  an  etiologic  factor  in  the  disease.  For,  in  a  case  of  this  kind,  it  is 
impossible  to  be  absolutely  sure  that  the  individual  has  never  had 
the  disease,  and  adults  at  this  age  may  be  immune.  The  result  does 
indicate  surely  that  this  organism,  present  almost  constantly  as  it  is 
in  pertussis,  and  capable  of  producing  such  reaction  in  an  adult, 
must  be  of  some  significance  in  the  disease,  and  is  at  least  not  always 
a  harmless  saprophyte. 

V.  INFLUENZA-LIKE  ORGANISMS  IN  OTHER  DISEASES  AND  IN  NORMAL 

THROATS. 

The  occurrence  in  other  diseases  of  what  appears  to  be  the  same 
organism,  morphologically  and  culturally,  is  of  interest.  A  number 
of  examinations  were  made  of  sputum  and  throat  swab  by  the  same 
method  as  that  used  in  whooping-cough,  from  cases  of  measles,  acute 
influenza,  epidemic  cerebrospinal  meningitis,  bronchitis,  varicella, 
and  from  normal  throats. 

Measles. — Twenty-two  cases  of  uncomplicated  measles  were 
examined.  The  sputum  was  obtained  in  most  of  the  cases,  but  in  a 
few  this  was  impossible,  and  throat  swabs  were  used.  All  were 
examined  during  the  stage  of  eruption,  and  most  of  them  had  at  the 
time  the  usual  cough  present  early  in  the  disease.  Only  one  exami- 
nation was  made  in  each  case.  The  influenza-like  bacilli  were  iso- 
lated in  pure  culture  in  13  of  the  22  cases.  In  four  of  the  cases  they 
were  the  predominating  organism ;  in  some  cases  they  were  very  few 


26  David  J.  Davis 

in  number.  Streptococci  and  pneumococci  were  found  in  every 
plate,  the  latter  always  predominating.  The  M.  cat arr halts  was 
found  in  lo  of  the  cases,  being  extremely  abundant  in  two.  Diph- 
theria bacilli  were  obtained  from  the  throats  in  two  cases.  They 
were  large  curved  bacilli  containing  large  granules,  and  the  col- 
onies showed  a  distinct,  clear  zone  of  hemolysis.  One  of  the 
cases  had  a  profuse  nasal  discharge  during  convalescence.  In  this 
material  were  found  by  culture  pneumococci,  streptococci,  a  few 
diphtheria  bacilh  and  a  considerable  number  of  influenza-like  bacilli. 
Four  of  the  cases  were  complicated  with  otitis  media,  and  the  pus 
was  examined  in  each  case.  In  one  case  the  influenza-like  bacilli 
were  obtained  nearly  pure,  there  being  present  also  a  few  strepto- 
cocci. In  this  case  the  bacilli  had  been  obtained  from  the  throat 
about  a  week  previously;  ii  days  later  the  pus  was  again  examined, 
and  showed  no  influenza  bacilli,  but  nearly  a  pure  culture  of  a  bacillus 
of  the  diphtheria  group.  From  the  other  three  cases  streptococci 
were  obtained,  pure  in  one  of  them,  and  streptococci  and  diphtheria 
bacilli,  mixed,  in  the  remaining  two. 

Clinical  influenza  or  grippe. — The  sputum  was  examined  from 
17  cases  that  were  diagnosed  clinically  as  acute  influenza.  They 
occurred  at  a  time  when  there  was  reported  to  be  an  epidemic  of 
grippe  in  the  city..  In  only  three  cases  were  the  influenza  bacilli 
found,  and  in  only  one  of  these  were  they  very  numerous.  Strepto- 
cocci were  very  conspicuous  in  these  cases,  and  while  not  so  numerous 
as  pneumococci,  they  were  more  abundant  than  in  the  other  groups 
examined. 

Epidemic  cerebrospinal  meningitis. — In  five  cases  of  typical 
cerebrospinal  meningitis  the  influenza-like  bacilli  were  found  either 
in  the  nose  or  throat  of  four.  The  case  in  which  it  was  not  found 
had  been  sick  for  three  weeks  when  examined,  and  at  that  time 
showed  no  nasal  or  throat  symptoms.  The  other  four  cases  were 
examined  early  in  the  disease,  and  showed  some  nasal  and  throat 
symptoms.  In  one  of  these  the  meningococcus  was  isolated  from 
the  nasal  cavity  and  also  the  sputum,  and  in  this  same  case  the 
influenza-like  bacilli  were  also  present  in  large  numbers,  being  by 
far  the  predominating  organism.  The  meningococcus  was  culti- 
vated from  the  cerebrospinal  fluid  in  each  of  the  five  cases. 


Bacteriology  of  Whooping- Cough  27 

Bronchitis. — This  comprises  a  more  or  less  miscellaneous  group 
of  cases.  All  had  bronchitis,  some  for  a  long  period,  while  in  others 
it  occurred  in  a  more  acute  condition.  In  some  cases  it  was  a  com- 
plication of  some  other  disease.  The  influenza  bacilli  were  found 
in  five  of  the  12  cases  examined.  In  two  of  these  cases  they  were 
very  numerous,  by  far  exceeding  all  other  organisms  present.  One 
case  in  this  series  was  a  typhoid  which  began  with  a  marked  bron- 
chitis. Here  was  found,  in  practically  pure  culture,  the  M.  catar- 
rhalis,  but  no  influenza-like  organisms.  This  micrococcus  was 
found  in  only  one  other  case  of  this  group. 

Varicella. — Throat  swabs  from  11  cases  of  varicella  in  young 
children  were  examined  in  the  usual  way.  All  of  the  children  had 
a  slight  cough,  and  three  had  at  the  time  of  the  examination  a  gonor- 
rheal vaginitis.  The  influenza-like  bacilli  were  isolated  in  seven  of 
the  eleven  cases;  in  two  they  were  numerous.  Streptococci  were 
found  in  eight, .  diphtheria  bacilli  in  one,  and  the  M.  catarrhalis 
in  all  of  the  cases.  The  latter  organism  was  very  numerous  in  two 
cases  in  which  the  influenza-like  organisms  were  absent. 

Normal  throats. — For  these  examinations  swabs  were  made  from 
the  posterior  wall  of  the  pharynx  and  were  for  the  most  part  obtained 
from  medical  students.  In  20  cases  the  influenza-like  bacilli  were 
found  twice;  in  neither  case  were  they  numerous.  The  throats  of 
these  two  individuals  appeared  normal,  and  they  gave  no  history  of 
any  throat  trouble  for  at  least  three  months  previously,  remaining 
perfectly  well  for  several  weeks  at  least  following  the  examination. 

Table  2  gives  a  summary  of  the  occurrence  of  influenza-like  bacilli 
in  the  diseases  investigated.  It  should  be  noted  that  in  all  the  diseases 
except  whooping-cough  but  one  examination  was  made.  In  this 
disease,  in  a  number  of  cases,  several  examinations  were  made  before 
finding  the  bacilH.  It  is  therefore  quite  probable  that  if  more  exami- 
nations were  made  in  the  other  conditions  the  percentage  of  positive 
results  would  have  been  higher. 

The  bacilli  from  these  various  sources  were  carefully  observed 
and  their  properties  tested.  Every  organism  was  transplanted  to 
non-hemoglobin  media,  and  not  one  was  found  which  would  grow. 
Particular  attention  was  given  to  the  test  on  ascites-agar  and  serum- 
agar,  as  so  many  reports  have  been  made  that  organisms  of  this  kind 


28 


David  J.  Davis 


TABLE   2. 
Occurrence  of  Influenza-like  Bacilli  in  Various  Diseases  and  in  Normal  Throats 


No.  of 

Cases 

Examined 

Influenza- 
like Bacilli 
Present 

Influenza- 
like Bacilli 
Not  Found 

Percentage 
of  Positive 
Findings 

Whooping-cough 

Epidemic  cerebro- 
spinal meningitis 

Varicella 

6i 

5 
II 

22 
12 

17 

20 

S6 

4 
7 
13 
S 
3 

2 

5 

I 
5 
9 
7 
14 
i8 

92 

8o 
63 
59 
42 
18 

Measles    . . . 

Influenza  (grippe) 

occur  which  will  develop  upon  these  media.  In  no  instance  did  a 
continuous  growth  occur  when  proper  precautions  were  taken  to  use 
fluids  free  from  hemoglobin.  Their  cultural  characteristics  were 
identical,  and  morphologically  there  were  no  constant  differences 
between  them.  In  every  group  strains  were  found  with  a  tendency 
to  marked  thread  formation,  and  there  was  also  considerable  varia- 
tion in  the  size  of  the  various  strains,  but  nothing  was  observed  that 
would  characterize  any  particular  group.  Animal  experiments 
showed  that  the  bacilli  from  the  different  groups  possessed  about  the 
same  pathogenic  power  as  those  isolated  from  whooping-cough  cases. 
Even  those  isolated  from  normal  throats  manifested  the  same  low 
degree  of  virulence  as  the  other  strains.  A  large  quantity  of  the 
bacilli  is  necessary  to  produce  death,  and  the  symptoms  are  not  those 
of  a  toxemia  but  of  a  general  invasion,  with  numerous  bacilh  in  the 
blood. 

Literature. — In  the  literature  there  are  numerous  references  to 
the  occurrence  of  hemophilous  bacilli  in  many  infectious  diseases 
and  diseases  of  the  respiratory  tract.  They  have  usually  been  referred 
to  as  influenza  bacilh  (Pfeiffer's  bacillus),  occasionally  as  pseudo- 
influenza  organisms,  and  for  the  most  part  have  been  looked 
upon  as  secondary  invaders,  except  in  influenza,  in  which  the 
hemophilous  bacilli  have  pretty  generally  come  to  be  considered 
as  the  specific  organism.  Pfeiffer,  in  1893,  in  his  classical  work 
on  influenza,  was  the  first  to  announce  this,  and  since  then  an 
enormous  mass  of  hterature  has  accumulated,  most  of  which  tends 
to  substantiate  the  idea  that  his  baciflus  is  the  specific  cause  of  this 
disease.     If  the  evidence  is  carefully  examined,  however,  it  is  found 


Bacteriology  of  Whooping-Cough  29 

that  in  reality  the  specificity  of  this  organism  does  not  rest  upon  any 
too  sure  foundation.  It  does  not  meet  Koch's  requisites.  In  many 
epidemics  the  bacilli  are  constant  in  the  throats,  being  often  present 
in  large  numbers;  and  they  have  been  obtained  from  abscesses  and 
numerous  other  lesions  in  the  body.  In  postmortems  the  organisms 
are  found  in  large  numbers,  especially  in  the  respiratory  passages 
and  the  lungs.  It  is  somewhat  questionable  whether  the  influenza 
bacillus  has  ever  been  obtained  from  the  blood  during  life.  Canon's 
results  are  absolutely  discredited  by  Pfeiffer  himself,  and  the  work 
of  Jehle,  Isambert,  Rosenthal,  and  others  needs  further  confirmation. 
The  pathogenicity  of  the  bacilli  for  animals  is  very  low,  and  the 
experiments  of  Pfeiffer  in  regard  to  the  presence  of  toxins  are  not  at 
all  convincing — though  the  more  recent  work  of  KoUe  and  Delius  and 
Slatineano  indicate  their  occurrence.  These  facts  are  hardly  suffi- 
cient to  demonstrate  beyond  question  that  the  organism  is  specific, 
and  that  it  is  not  merely  a  secondary  invader. 

It  is  interesting  that  the  French  especially  have  always  been  skeptical  about  the 
etiologic  r61e  of  Pfeiffer's  bacillus  in  influenza;  and  many  data  have  been  presented 
in  opposition  to  the  view  that  it  is  specific  for  this  disease.  Elmassian,'  in  1899,  could 
not  distinguish  between  organisms  obtained  from  whooping-cough,  acute  bronchitis, 
pulmonary  tuberculosis,  and  pneumonia.  He  considered  them  all  identical  with 
Pfeififer's  bacillus  of  influenza,  and  said  that  it  was  not  proved  that  this  bacillus  was 
the  specific  cause  of  grippe,  but  should  be  considered  only  the  probable  cause.  Rosen- 
thal2  quotes  MetchnikofF  as  saying  that  Pfeiff"er  could  not  find  the  influenza  organisms 
in  an  epidemic  of  grippe  in  1899.  He  thinks  it  an  ordinary  microbe  of  the  pathologic 
flora  of  the  lungs,  and  not  specific  for  grippe.  Sacquepee3  studied  a  typical  and  very 
severe  epidemic  of  grippe  in  a  garrison,  and  found  Pfeiffer's  bacillus  in  only  the  latter 
part  of  the  epidemic  (February).  At  other  times  he  found  a  Gram-negative,  motile, 
typhoid-like  bacillus,  and  also  pneumococci  and  streptococci.  He  thinks  that  the 
influenza  bacillus  is  not  specific,  and  that  grippe  may  not  be  due  to  any  specific  microbe* 
Bezan9on  and  Israels  de  Jong,*  in  an  epidemic  of  grippe,  studied  the  expectorations  of 
25  cases  bacteriologically,  and  found  Pfeiffer's  bacillus  to  be  very  rare.  They  call 
special  attention  to  "Micrococcus  catarrhalis"  and  to  " paratetragene  zoogleique" 
which  were  very  common.  They  found  pneumococci,  streptococci,  pseudo-diphtheria, 
pneumobacilli,  and  rarely  staphylococci.  They  conclude  that  grippe  is  not  caused  by 
Pfeiffer's  bacillus,  but  is  due  to  a  variety  of  organisms  of  exalted  virulence.  Kleinen- 
berger,s  in  27  cases  of  epidemic  influenza,  found  the  influenza  bacillus  only  eight 
times.     The  M.  catarrhais  was  present  in  nearly  every  case.      Lord,^  of  Boston,  has 

'  Ann.  de  I' Inst.  Past.,  1899,  13,  p.  621. 

»  Compt.  rend,  de  la  Soc.  de  Biol.,  1900,  52,  p.  266. 

3  Arch,  de  mid.  expir.,  1901,  13,  p.  562. 

•♦  Reviewed,  Bull,  de  I'Inst.  Past.,  1905,  3,  p.  372. 

i  Deutsche  med.  Wchnschr.,  igos   31,  p.  575. 

^Boston  Med.  and  Surg.  Jour.,  1905,  152,  pp.  537  and  574. 


30  David  J.  Davis 

studied  non-epidemic  infections  of  the  respiratory  tract,  and  has  clearly  shown  the 
similarity,  both  clinically  and  pathologically,  of  the  infections  due  to  influenza  bacilli 
and  other  organisms,  such  as  pneumococcus  and  M.  catarrhalis.  Von  Jaksch^  has 
described  a  condition,  under  the  name  of  pseudo-influenza,  which  is  indistinguishable 
clinically  from  influenza,  but  which  is  not  caused  by  the  influenza  bacillus.  He 
concluded  that  it  was  due  often  to  a  streptococcus  invasion,  because  of  the  large 
number  of  these  organisms  found;  in  some  the  cause  could  not  be  determined,  but 
he  was  sure  it  was  not  due  to  influenza  infection.  2 

The  results  of  these  investigations  correspond  very  well  with  the 
findings  in  the  series  of  influenza  cases  reported  in  this  paper.  While 
it  is  undoubtedly  true  that  in  many  epidemics  of  influenza  Pfeiffer's 
bacillus  exists  constantly  in  the  nasal  and  oral  excretions  in  very 
large  numbers,  and  to  the  exclusion  largely  of  other  bacteria,  and 
that  its  presence  corresponds  with  the  course  of  the  disease,  it  is 
equally  true  that  there  occur  epidemics,  and  also  sporadic  cases,  of 
a  condition  which  appears  to  be  identical  with  influenza  clinically, 
but  which  shows  Pfeiffer's  bacillus  only  occasionally,  and  other 
organisms,  such  as  pneumococci,  staphylococci,  and  M.  catarrhalis, 
very  commonly  and  in  large  numbers.  The  most  rational  interpreta- 
tion of  these  facts  seems  to  be  that  there  are  a  number  of  organisms 
which  may  give  clinically  the  same  picture,  and  which  may  exist 
in  the  respiratory  passages  either  in  nearly  pure  culture;  or  they 
may  occur  together. 

The  fact  that  many  cases,  indistinguishable  clinically  from  acute 
influenza,  occur  both  in  sporadic  and  epidemic  form  not  associated 
with  the  influenza  bacillus,  should  be  more  widely  known  among 
the  medical  profession  at  large.  For  ever  since  the  great  epidemic 
of  1889-90,  almost  everything  bearing  any  resemblance  at  all  to 
influenza  is  assumed,  without  a  bacteriologic  examination,  to  be  an 
influenza  infection.  Attention  should  be  called  also  to  the  fact  that 
smears  of  oral  and  nasal  excretions  are  very  unrehable  in  the  diagno- 
sis of  this  disease,  and  that,  to  be  sure  of  an  influenza  infection, 
cultures  on  blood-agar  plates  should  be  made.     Chnical  data,  there- 

'  Berl.  kliii.  Wchnschr.,  1899,  36,  p.  425. 

»  Since  this  paper  was  written  an  article  upon  influenza  infections  by  Jochmann  {Deuisches  Arch 
f.  klin.  Med.,  1905,  84,  p.  470)  has  appeared.  In  36  cases  of  epidemic  enfluenzas  he  was  able  to  find 
influenza  bacilli  in  only  13.  Pneumococci  and  streptococci  were  very  common.  He  thinks  that  the 
clinical  picture  of  acute  influenza  may  be  produced  by  organisms  other  than  the  influenza  bacilli; 
especially  the  pneumococcus,  streptococcus,  and  perhaps  the  M.  catarrhalis.  He  discusses  the  occur- 
rences of  influenza-like  bacilli  in  the  infectious  diseases  and  says  the  organisms  from  whooping-cough 
are  indistinguishable  morphologically  and  biologically  from  the  bacillus  of  Pfeiffer.  His  results  agree 
very  well  with  those  given  in  this  paper. 


Bacteriology  of  Whooping- Cough  31 

fore,  unless  accompanied  by  careful  bacteriological  examinations, 
are  of  little  value.  This  is  mentioned  because  in  the  literature,  even 
of  late  years,  so  much  is  written  upon  influenza  without  definite 
knowledge  of  the  presence  of  influenza  bacilH. 

Besides  influenza,  in  which  hemophilous  bacilli  occur,  and  are 
the  probable,  but  not  the  demonstrated,  cause  of  the  disease,  there 
are  numerous  other  diseases  in  which  hemophilous  bacilli,  usually 
called  influenza  or  pseudo-influenza  organisms,  have  been  frequently 
found.  A  brief  resume  of  the  more  important  literature  upon  this 
point  will  here  be  given. 

Pfeiflfer,  as  already  stated,  found  what  he  called  pseudo-influenza  bacilli  in  broncho- 
pneumonia, and  distinguished  them  from  influenza  by  their  greater  size  and  their 
tendency  to  form  threads.  Numerous  writers  since  that  time  have  noted  the  marked 
tendency  to  form  threads  in  bacilli  from  cases  of  influenza,  and  have  also  noted 
considerable  variation  in  size  in  various  strains  of  such  organisms.  Also,  attention 
has  been  called  to  the  occurrence  in  other  conditions  of  bacilli  not  differing  morphologi- 
cally from  Pfeifi"er's  influenza  bacillus.  Pfeiffer's  distinction,  therefore,  between  the 
bacilli  from  influenza  cases  and  those  from  other  sources  does  not  appear  to  hold  true, 
and  the  pseudo-influenza,  for  the  present,  must  be  considered  identical  with  the  influ- 
enza bacillus.  Elmassian  found  influenza  bacilli  in  acute  bronchitis,  tuberculosis, 
and  pneumonia.  Rosenthal^  found  them  in  15  out  of  19  cases  of  broncho-pneumonia, 
nearly  pure  in  two.  Susswein^  obtained  them  in  10  out  of  21  cases  of  measles.  Lo  •d,3 
in  a  very  valuable  paper,  reports  his  findings  in  diseases  of  the  respiratory  tract,  chiefly 
acute  and  chronic  bronchitis.  He  obtained  influenza  bacilU  in  every  way  correspond- 
ing to  Pfeiff'er's  bacillus  in  56  out  of  186  cases  (30  per  cent),  and  in  47  cases  the  bacilli 
were  in  overwhelming  numbers.  These  cases  were  examined  at  a  time  when  there 
was  prevalent  no  epidemic  of  any  acute  respiratory  disease.  Smith*  found  influenza 
bacilli  five  times  in  73  cases  of  lobar  pneumonia.  LeinerS  obtained  them  constantly 
from  the  bronchial  secretion  and  lungs  of  1 1  postmortem  cases  of  diphtheria.  Neisser^ 
found  them  in  cases  of  measles,  scarlet  fever,  and  whooping-cough.  Jehle?  found 
influenza  bacilli  in  postmortems  as  follows:  In  48  cases  of  scarlatina,  ig  times  in  the 
lungs,  six  times  in  the  tonsils,  and  22  times  in  the  blood;  in  nine  cases  of  varicella, 
nine  times  in  the  lungs  and  five  times  in  the  blood;  in  24  cases  of  pertussis,  24  times  in 
the  lungs  and  12  times  in  the  blood;  in  15  cases  of  diphtheria,  nine  times  in  the  lungs 
and  five  times  in  the  blood.  Liebscher,*  in  57  cases  of  measles,  found  influenza 
bacilli  1 1  times,  and  in  60  cases  of  scarlatina  found  them  three  times.  He  examined  only 
the  nasal  secretions.  Auerbacho  found,  in  swabs  from  the  tonsils  and  larynx  of  700 
cases  of  acute  infectious  diseases,  the  influenza  bacillus  12  times  in  diphtheria,  three 
times  in  scarlatina,  six  times  in  what  he  calls  diphtheria-scarlatina,  seven  times  in 
diphtheria-morbilli,   and    10  times  in  anginas.     Kleinenberger^o  found  hemophilous 

'  Compt.  rend,  de  la  Soc.  Biol.,  1900,  52,  p.  226.  *  Deutsche  med.  Wchnschr.,  1903,  29,  p.  462. 

»  Wiener  hlin.  Wchnschr.,  1901,  14,  p.  ii49-  '  Ztschr.  /.  Heilkunde,  1901,  22,  p.  190. 

3  Brit.  Med.  and  Surg.  Jour.,  1905,  152,  p.  537-  *  Prager  med.  Wchnschr.,  1903,  28,  p.  90- 

4  .Jour.  Boston  Soc.  of  Med.  Sc,  3,  p.  274-  '  Ztschr.  /.  Hyg.,  1905,  47.  P-  259- 

s  Wiener  klin.  Wchnschr.,  1901,  14,  p.  looi.  ^"Deutsche  med.  Wchnschr.,  1905,  31.  P-  575 


32  David  J.  Davis 

bacilli  in  i8  out  of  25  cases  of  pertussis,  upon  the  conjunctiva  in  three  out  of  nine  cases 
of  measles,  once  in  the  heart's  blood  in  measles  postmortem,  once  in  scarlet  fever  and 
once  in  a  case  of  urethritis.  So  far  as  I  have  been  able  to  determine,  there  are  no 
reports  in  the  literature  upon  the  occurrence  of  influenza  bacilli  in  normal  throats. 

To  sum  up  the  literature,  the  results  agree  very  well  with  the 
data  given  in  Table  2,  so  far  as  they  are  comparable.  Siisswein's 
results  in  measles  and  Lord's  results  in  bronchitis  correspond  very 
closely  with  those  in  the  table.  The  findings  in  influenza  agree  in 
general  with  those  obtained  by  many  investigators,  especially  the 
French.  I  do  not  know  of  any  positive  findings  in  the  literature  of 
influenza  bacilli  in  the  nose  or  throats  of  cases  of  epidemic  meningitis. 
All  the  results  taken  together  indicate  that  we  have  in  these  bacilli 
an  organism  occurring  very  commonly  in  a  large  number  of  diseases, 
and  undoubtedly  present,  at  times  at  least,  in  all  inflammatory  con- 
ditions of  the  respiratory  passages  whatever  their  character  may  be. 
The  evidence  is  conclusive  that  this  organism  at  times  exists  as  a 
saprophyte,  as  indicated  by  its  appearance  in  normal  throats,  and 
that  it  is  also  capable  of  causing  inflammatory  changes,  as  shown 
both  by  human  and  by  animal  experiments,  and  by  the  vast  amount 
of  pathologic  data.  The  all-important  question  is,  whether  we  are 
dealing  in  all  these  various  conditions  with  the  same  organism, 
varying  only  in  its  degree  of  virulence,  or  whether  we  have  to  do  here 
with  a  group  of  very  closely  related  organisms.  Morphologically, 
tinctorially,  and  culturally,  from  the  results  obtained  from  the  various 
strains  isolated  in  the  cases  here  reported,  and  in  the  cases  reported 
in  the  literature,  we  must  conclude  that  we  have  no  means  of  dis- 
tinguishing them.  Reasoning  from  analogy,  we  should  expect  a 
group,  as  occurs  with  so  many  of  the  other  bacteria;  and  the  chief 
problem  now  before  us  is  the  careful  application  of  every  possible 
means,  particularly  in  the  way  of  biological  experiments,  to  differ- 
entiate the  organisms.  Already  considerable  evidence  from  this 
point  of  view  exists,  but  it  is  conflicting.  Wollstein's  experiments 
upon  agglutination  with  the  organisms  from  influenza  and  pertussis 
are  extremely  important  and  suggestive.  They  are  in  accord  with 
results  obtained  by  Cantani^  with  the  influenza  bacillus,  but  are 
opposed,  directly  or  indirectly,  to  the  results  obtained  by  several 
investigators  (Neisser,  Smit,  Lord,  Jehle,  Meunier). 

'  Ztschr.  f.  Hyg.,  1903,  42,  p.  505. 


Bacteriology  of  Whooping -Cough  33 

It  is  perhaps  worth  while  at  this  time  to  call  attention  to  the  fact 
already  noted  by  Jochmann,  that  whatever  may  be  said  in  favor  of 
the  influenza  bacillus  as  the  cause  of  influenza,  practically  the  same 
thing  may  be  said  in  favor  of  the  organism  isolated  from  the  cases 
of  pertussis  as  the  cause  of  that  disease.  The  constant  occurrence 
of  the  organism  at  the  apparent  seat  of  disease  in  practically  all 
uncomplicated  cases,  the  parallelism  between  the  general  course  of 
the  disease  and  the  relative  abundance  of  the  organisms,  the  presence 
of  the  organisms  in  the  lungs  and  various  secretions  in  postmortems, 
the  low  pathogenicity  of  the  organism  for  animals — all  these  hold 
true  for  one  equally  as  well  as  for  the  other.  The  medical  world, 
with  few  exceptions,  has  come  to  recognize  the  influenza  organism 
as  specific  for  this  disease,  but  it  surely  is  not  yet  ready  to  accept  the 
cause  of  whooping-cough  as  settled.  This  fact  may  be  interpreted 
in  two  ways ;  it  may  be  used  to  indicate  upon  how  insecure  a  founda- 
tion the  specificity  of  the  influenza  bacillus  rests,  or  it  may  be  used 
as  an  argument  in  favor  of  the  specificity  of  the  pertussis  organism. 
Evidently,  with  the  present  data  at  hand,  it  is  impossible  to  settle 
either  question  absolutely. 

Pertussoid,  pseudo- pertussis,  etc. — A  few  words  may  be  said  con- 
cerning a  condition  variously  referred  to  in  the  literature  as  a 
whooping-cough-Hke  cough  in  influenza,  pertussoid,  pseudo-pertussis, 
etc.,  by  Leichtenstern,  Forchheimer,  Filatow,  Guidi,  Pestalozza  and 
others.  From  the  description  given,  it  would  appear  that  these  con- 
ditions are  intermediary  forms  between  influenza  on  the  one  hand,  and 
whooping-cough  on  the  other.  This  is  certainly  suggestive  in  view 
of  the  fact  that  bacilli  so  nearly  alike,  if  not  identical,  are  found  so 
constantly  in  both  these  diseases.  Forchheimer^  was  able  to  find 
the  influenza  bacillus  in  but  few  of  the  cases,  but  found  strepto- 
cocci constantly. 

I  have  had  an  opportunity  of  examining  only  two  cases  which 
might  come  under  this  class.  Both  were  adults,  and  both  had  come 
in  contact  more  or  less  with  whooping-cough  patients.  Each  had  a 
very  severe  cough,  lasting  several  weeks,  spasmodic  in  character  but 
without  distinct  whoops.  In  the  sputum  of  both,  influenza-like 
bacilli  were  found,  quite  numerous  in  one,  but  very  few  in  the  other. 

•  Archives  of  Pediatrics,  1900,  17,  p.  8oi. 


34  David  J.  Davis 

In  the  former  were  also  many  streptococci,  while  in  the  latter  there 
was  nearly  a  pure  culture  of  pneumococcus.  A  careful  study,  with 
particular  reference  to  the  influenza-like  bacilli,  should  be  made  of 
the  flora  of  the  oral  and  nasal  secretions  of  a  large  series  of  such 
cases,  and  especially  those  cases  of  cough  occurring  in  parents  or 
persons  associated  with  children  having  pertussis,  and  often  referred 
to  as  a  sympathetic  cough,  and  the  like. 

VI.      NOMENCLATURE   OF  THE   BACILLI. 

Before  concluding,  it  may  be  well  to  discuss  briefly  the  nomen- 
clature of  the  bacilli  of  this  class.  As  a  general  term  for  organisms 
of  this  type,  which  only  grow  upon  hemoglobin  media,  "Bacillus 
hemophilicus,^^  used  so  generally  by  the  French,  certainly  seems 
appropriate,  inasmuch  as  it  describes  their  most  important  char- 
acteristic. Such  a  term  would  include  Pfeiffer's  bacillus,  the  pseudo- 
influenza  bacillus,  all  the  bacilli  described  as  influenza-like  or  as 
influenza  bacilli  found  in  whooping-cough,  measles,  scarlet  fever, 
diphtheria,  pharyngitis,  varicella,  bronchitis,  tuberculosis,  etc.,  Miiller's 
trachoma  bacillus,  and  a  few  other  organisms  having  this  property  and 
isolated  from  various  sources.  Should  differences  be  detected  between 
the  various  organisms,  as  in  all  probability  there  will  be,  at  least 
between  some  of  them,  special  names  may  then  be  assigned  them ;  this 
has  been  done,  by  those  who  believe  that  there  are  detectable  differ- 
ences, with  organisms  from  influenza  and  whooping-cough.  As  for  the 
last- mentioned  organism,  if  the  names  of  individuals  are  used  to  desig- 
nate it,  it  should  be  known  either  as  Spengler's  bacillus  or  the  Speng- 
ler-Jochmann  bacillus.  After  reading  Spengler's  description  of  this 
organism,  it  is  difficult  to  see  how  anyone  can  believe  that  he  was 
dealing  with  an  organism  different  from  that  described  by  Jochmann, 
as  the  latter  writer  contends.  Spengler  called  this  organism  the 
pertussis  bacillus,  and  if  this  organism  is  proved  beyond  question  to 
be  specific  for  the  disease  and  different  from  B.  influenzae,  this  term 
should  be  used,  and  Jochmann's  term,  "Bacillus  pertussis  Eppen- 
dorf,"  discarded.  However,  until  these  organisms  are  definitely 
distinguished  from  each  other,  it  would  seem  that  the  special  term, 
Bacillns  influenzae,  should  be  used  for  all. 


Bacteriology  of  Whooping- Cough 


VII.      SUMMARY   AND  CONCLUSIONS. 


35 


1.  In  the  sputum  of  pertussis  patients  there  is,  almost  constantly, 
an  organism  which,  morphologically  and  culturally,  is  identical  with 
the  influenza  bacillus.  It  is  usually  in  greater  abundance  than  any 
other  organism  of  the  sputum. 

2.  This  organism  is  most  abundant  during  the  spasmodic  stage  of 
the  disease.  It  has  been  found  several  days  before  the  whoops 
began,  and  as  long  as  six  months  after  the  disease. 

3.  During  the  coughing  spasms  it  is  thrown  out  in  the  surrounding 
air  in  large  numbers. 

4.  Alone,  its  pathogenicity  for  animals  is  low;  when  associated 
with  other  organisms,  this  property  is  decidedly  increased. 

5.  When  transplanted  in  the  human  throat,  this  organism  grows 
in  abundance  and  gives  rise  to  a  distinct  reaction.  It  is  therefore 
not  a  harmless  saprophyte. 

6.  Symbiotic  phenomena  are  manifested  by  this  organism  exactly 
as  by  Pfeiffer's  bacillus. 

7.  Organisms  which  have  not  been  differentiated  from  this  bacillus 
occur  in  a  variety  of  throat  affections,  and  occasionally  in  normal 
throats. 

8.  The  evidence  at  hand  will  not  permit  a  definite  statement  for 
or  against  the  specificity  of  this  organism  for  whooping-cough. 

9.  Granting  that  it  is  not  specific,  its  significance  as  a  harmful 
secondary  invader  cannot  be  questioned. 

10.  This  organism  was  described  first  by  Spengler  in  1897,  and 
later  by  Jochmann,  Krause,  and  others.  Undoubtedly  many  have 
observed  it,  but  failed  to  isolate  or  describe  it  properly. 

I  wish  to  acknowledge  my  indebtedness  to  Dr.  Hektoen  for  sug- 
gestions and  to  Drs.  Baum  and  Weaver,  attending  physicians  at 
Cook  County  Hospital,  for  the  privilege  of  utiHzing  material  in  the 
infectious  wards.  I  wish  also  to  thank  the  internes  at  Cook  County 
Hospital  for  their  many  courtesies,  and  also  numerous  physicians 
who  aided  me  in  obtaining  material  for  examination. 

EXPLANATION   OF  PLATE  I. 
Fig.  I. — Symbiosis — Cluster  of  colonies  of  the  influenza-like  bacillus  around  the 
larger    staphylococcus   colony.      Blood-agar   plate  inoculated  directly  from  pertussis 
sputum.     Magnified  30  times. 


36  David  J.  Davis 

Fig.  2. — Influenza-like  bacilli  from  24  hour  blood-agar  culture  isolated  from 
pertussis  sputum.    Some  of  the  bacilli  show  polar  staining.    Magnified  1,200  diameters. 

Fig.  3. — Phagocytosis  of  influenza-like  bacilli  in  sputum  of  pertussis  patient. 
Magnified  1,200  diameters. 

Fig.  4. — Twenty-four  cultures  of  influenza-like  bacilli  from  pertussis  sputum 
showing  marked  tendency  to  thread  formation.     Magnified  1,200  diameters. 

Fig.  5. — Influenza-like  bacilli  in  smear  of  sputum  from  case  inoculated  with 
these  organisms.     Magnified  1,200  diameters. 

Fig.  6. — Phagocytosis  of  leucocytes  by  endothelial  cells  seen  in  peritoneal  exudate 
of  guinea-pig  injected  with  the  influenza-like  bacilli.     Magnified  1,200  diameters. 


PLATE  I. 


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Fig.  2. 


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Fig.  3. 


Fig.  4. 


vv 


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i 


Fig.  6. 


Or 


rH: 


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!iR201     Da^is,   D.    J.  $^9469 

W6D2  ^The  bacteriology  of  ifhoop. 

1906  ing- coughs 


